Therapeutic methods and compositions involving allosteric kinase inhibition

ABSTRACT

The present invention is directed to methods and compositions for suppressing lymphangiogenesis, angiogenesis and/or tumor growth. The methods comprise contacting the tumor with a compound that (i) stabilizes a protein kinase in the inactive state and (ii) is not an ATP competitive inhibitor of the protein kinase in the active state.

CROSS-REFERENCE

This application is a division of U.S. patent application Ser. No.13/577,431, filed Dec. 5, 2013, which claims priority to U.S.International Patent Application No. PCT/US2011/023949, filed Feb. 7,2011, which claims the benefit of U.S. Provisional Application No.61/302,471, filed Feb. 8, 2010, and U.S. Provisional Application No.61/310,663, filed Mar. 4, 2010, which applications are incorporatedherein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentby grants from the NIH: P01-CA078045 (DAC), P01-HL057900 (DAC), andP01-CA104898 (DAC).

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 2, 2016, isnamed 42242-801.401_SL.txt and is 10,383 bytes in size.

BACKGROUND OF THE INVENTION

Kinases regulate fundamental processes in cancer including tumorproliferation, metastasis, neovascularization, and chemoresistance.Accordingly, kinase inhibitors have been a major focus of drugdevelopment and several kinase inhibitors are now approved for variouscancer indications. Typically, kinase inhibitors are selected via highthroughput screening using catalytic kinase domains at low ATPconcentration and this process often yields ATP mimetics that lackspecificity and/or function poorly in cells where ATP levels are high.

SUMMARY OF THE INVENTION

In one aspect provides herein compounds having the structure

or an N-oxide, N,N′-dioxide, N,N′,N″-trioxide, or a pharmaceuticallyacceptable salt thereof, wherein R₁ and R₂ are independently hydrogen,optional substituted alkyl, halogen, optional substituted amine, NH₂,optional substituted alkyoxy, optional substituted thioalkyl, CF₃S,optional substituted alkylsulfinyl or optional substitutedalkylsulfonyl; Z′ is N or C; R^(m) is C₁₋₆ alkyl, or halogen substitutedC₁₋₆ alkyl; and R₃ is independently a hydrogen, C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, orC₃₋₁₀cycloalkyl; or, optionally, R^(m) and R₃ are joined to form a fiveto seven membered carbocycle; and n is 0-4.

In another aspect, there are provided processes for preparing a compoundof formula I:

comprising reacting a compound of formula II,

with a compound of formula (III),

in the presence of a base, wherein W is selected from the groupconsisting of pyridine, pyridazine, pyrimidine, pyrazine, triazine,quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline,1,2,4-benzotriazine and azaindole; X is O, S or NH; R₁ and R₂ areindependently a hydrogen, optional substituted alkyl, halogen, optionalsubstituted amine, —NH₂, —OH, optional substituted alkoxy, optionalsubstituted thioalkyl, —SCF₃, optional substituted alkylsulfinyl or anoptional substituted alkylsulfonyl; R₃ is independently a hydrogen,halogen substituted C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkoxy and mis 1-5.

In another aspect, there are provided processes for preparing a compoundof formula Ia:

comprising reacting a compound of formula IIa,

with a compound of formula (IIIa),

in the presence of a base, wherein R₁ and R₂ are independently ahydrogen, optional substituted alkyl, halogen, optional substitutedamine, —NH₂, —OH, optional substituted alkoxy, optional substitutedthioalkyl, —SCF₃, optional substituted alkylsulfinyl or an optionalsubstituted alkylsulfonyl and R₃ is a hydrogen, halogen, substitutedC₁₋₆ alkyl, or halogen substituted C₁₋₆ alkoxy.

In another aspect of the present invention, there are provided processesfor preparing a compound of formula IV:

comprising reacting a compound of formula V,

(V) with a compound of formula (VI),

in the presence of a base wherein L is a leaving group; Y is —SH, —OH,or —NH₂; R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted protected amine, optionalsubstituted alkoxy, optional substituted thioalkyl, CF₃S, optionalsubstituted alkylsulfinyl or optional substituted alkylsulfonyl and R₄is a C₁₋₆ alkyl.

Also provided herein are methods for lymphangiogenesis, angiogenesisand/or growth of a tumor. The methods comprise contacting the tumor witha compound that (i) stabilizing a protein kinase in the inactive stateand (ii) is not an ATP competitive inhibitor of the protein kinase inthe active state.

In another aspect, methods for treating cancer in a human subjectprovided herein comprise administering to a patient in need a compoundthat (i) stabilizes a protein kinase in the inactive state and (ii) isnot an ATP competitive inhibitor of the protein kinase in the activestate.

In another aspect, there are provided methods for preventing inhibitionof ASK1-mediated apoptosis in a cell comprising contacting the cell witha compound that (i) stabilizes the protein kinase in the inactive stateand (ii) is not an ATP competitive inhibitor of the protein kinase inthe active state.

In another aspect, methods for sensitizing a cell to an extrinsic stressprovided herein comprise contacting the cell with a compound that (i)stabilizes the protein kinase in the inactive state and (ii) is not anATP competitive inhibitor of the protein kinase in the active state.

In another aspect, there provided methods for inhibiting MEK1/2- and/orERK1/2-mediated cellular proliferation or migration comprisingcontacting a cell with a compound that (i) stabilizes the protein kinasein the inactive state and (ii) is not an ATP competitive inhibitor ofthe protein kinase in the active state.

In another aspect, there are provided methods for treating restenosis ina human subject comprising administering to a patient in need a compoundthat (i) stabilizes a protein kinase in the inactive state and (ii) isnot an ATP competitive inhibitor of the protein kinase in the activestate.

In another aspect, methods for treating fibrotic diseases in a humansubject provided herein comprise administering to a patient in need acompound that (i) stabilizes a protein kinase in the inactive state and(ii) is not an ATP competitive inhibitor of the protein kinase in theactive state.

In another aspect, there provided methods for inhibiting phosphorylationof S338 of CRAF and/or RAF dimerization comprising contacting a cellwith a compound that (i) stabilizes the protein kinase in the inactivestate and (ii) is not an ATP competitive inhibitor of the protein kinasein the active state.

The present invention also provides methods for identifying allostericinhibitors of RAF kinase. The methods comprise contacting a RAF kinasewith test compounds and monitoring the phosphorylation of S338 of CRAF,wherein a decrease in the phosphorylation of S338 of CRAF relative tonon-contacted RAF kinase indicates that the test compounds areallosteric inhibitor of RAF kinase.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference for the purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A illustrates the molecular modeling of the amino-triazole basedsmall molecules in PDGFRβ (right panel) and B-RAF (left panel). Thecrystal structure of B-RAF (PDB 1uwh) was selected for binding studiessince it contained the DFG motif of the activation loop in the desirableinactive state (i.e. DFG-out). A homology model of PDGFRβ was createdwith a structurally-related family member (VEGFR2, PDB 1y6b). Furtherdocking studies are provided in FIG. 6B.

FIG. 1B shows the human vascular smooth muscle cells (VSMC) pre-treatedwith compound 6 (0, 0.5, 1, and 10 μM) for 1 h followed by 7 minstimulation with PDGF-BB and lysis in RIPA buffer. Toautophosphorylation, immunoblots were carried out with aphospho-tyrosine antibody. Membranes were stripped and reprobed fortotal PDGFRβ levels.

FIG. 1C shows the Western analysis demonstrating compound 6 (5 μM)inhibition of bFGF or VEGF (50 ng/ml, 5 min stimulation) inducedphosphorylation of MEK and ERK in serum starved HUVECs. A comparison ofendothelial cell-based RAF inhibition of compounds 3 and 6 can be foundin FIG. 7A.

FIG. 1D shows the structure activity relationships of the amino-triazolebased small molecules comparing both PDGFRβ and B-RAF. Compounds 3through 11 were prepared (U.S. Pat. No. 7,652,051) and screened forinhibition of PDGF-BB induced PDGFRβ autophosphorylation in VSMCs at 2μM or bFGF induced MAPK activity in HUVECs at 5 μM by Western analysis(− denotes x<10%, + denotes 50%<x<70%, and ++ denotes x>90% inhibition).

FIG. 1E shows the VSMCs XTT viability curve in which VSMCs were treatedwith compounds 3, 6, imatinib, or sorafenib at various doses for 72 h infull growth medium containing 10% FBS. Curves represent the average of 3separate experiments, error bars represent ±SEM.

FIG. 1F shows the HUVECs XTT viability curve in which HUVECs weretreated with compounds 3, 6, imatinib, or sorafenib at various doses for72 h in full growth medium containing 10% FBS. Curves represent theaverage of 3 separate experiments, error bars represent ±SEM.

FIG. 2 provides illustrative profiles of compound 6, sorafenib, andimatinib in the KINOMEscan™ profiling service from Ambit Biosciences.The compounds were screened against 70 kinases for competitive bindingand the legend describes % control (inhibition relative to positivecontrol) of the kinase targets at 10 μM. The complete list of kinasesscreened as well as % inhibition is available in Table 2 and a list ofthe Kds of the kinase targets for compound 6 can be found in Table 3.

FIG. 3A illustrates the effect of compound 6 on angiogenesis intransgenic fli1-egfp zebrafish embryos. Embryos were treated with 1 μMcompound 6, SU5416, or 10 μM compound 3 from 20 hours post fertilization(hpf) until 48 hpf. Representative 3D reconstructions of the bloodvasculature are shown for both the head and tail regions of the embryo.Designations for the major vessels in the head and tail of the zebrafishembryo at 48 hpf are available in FIG. 8A.

FIG. 3B illustrates the disruption of lumen formation in the zebrafishduring late vessel maturation. Zebrafish treated with either 1 μMcompound 6 or 10 μM compound 3 from 20-48 hpf were imaged at 35 and 48hpf Bottom panel: compound 6 induces apoptosis in the intersegmentalvessels in the zebrafish. Shown is 3D overlap reconstruction of the GFPexpressing intersegmental vessels with TUNEL-positive nuclei at 48 hpf.Representative phase contrast views as well as quantification ofintersegmental vessel volumes from FIG. 3A and FIG. 3B can be found inFIG. 8B-D, respectively.

FIG. 3C illustrates the time course of drug addition to zebrafish.Compounds (2.5 μM for 3 or 6, 1 μM sorafenib, or DMSO as control) wereadded at either 18 hpf or 30 hpf (left on the embryos for the durationof the experiment) and intersegmental vessels were imaged approximately24 h after the time point corresponding to drug addition. At 30 hpf thezebrafish embryos established early vessels with evident lumens.Compound 6 and sorafenib were added to evaluate their effect onpre-existing vasculature.

FIG. 3D illustrates the phase contrast images of zebrafish treated withcompound 3, compound 6, or sorafenib (same concentrations as in FIG.3C). The images demonstrate that embryos treated with compound 3 andcompound 6 are viable whereas sorafenib causes death. n=4-6 zebrafishembryos per condition for all treatments. All scale bars=100 m.

FIG. 4A illustrates the effect of compound 3, compound 6, vatalanib,imatinib, GW 5074 and a combination of imatinib (PDGFR inhibitor) and GW5074 (RAF inhibitor) on intersegmental vessel formation inTg:(fli1-egfp) zebrafish embryos. Embryos were treated with DMSO, 5 μMcompound 3, 5 μM compound 6, 1 μM vatalanib, 5 μM imatinib (Im), 1 μM GW5074 (GW), and the combination of 5 μM imatinib and 1 μM GW 5074 (Im+GW)from 16 hours post fertilization (hpf) until 48 hpf. Z-stacks from laserscanning confocal microscopy are shown depicting formation of theintersegmental vessels at 48 hpf n=6 embryos/treatment.

FIG. 4B illustrates HUVECs co-cultured with hTERT-human hepatic stellatecells in a 3-D collagen matrix in the presence of complete EBM-2 mediumto monitor pericyte-associated endothelial tube formation. The stellatecells were labeled with 10 μg/ml red fluorescent dye (DiIC(3), BDBiosciences) for 1 h prior to the start of the experiment. Inhibitorswere added to the co-cultures 6 h post seeding at the followingconcentrations: DMSO, 2.5 μM 3, 2.5 μM compound 6, 1 μM imatinib (Im),0.5 μM GW 5074 (GW), and the combination of 1 μM imatinib and 0.5 μM GW5074 (Im+GW). The endothelial tubes were stained at 24 h by adding 2 μlFITC labeled Ulex europaeus lectin (Vector labs) per well. Images wereacquired 48 h post seeding of the cells. One representative panel from 3independent experiments is shown. Green—FITC lectin labeled endothelialcells, Red—DiIC(3) labeled stellate cells. Inset in each panel displaysa higher magnification view of the endothelial cell/stellate cellinteractions. Scale bar=200 m.

FIG. 4C illustrate tube lengths measured using Metamorph software foreach tube for all 10 fields that were acquired for compound 3, compound6, vatalanib, imatinib, GW 5074 and a combination of imatinib/GW 5074.The % pericyte-covered tube length was calculated from the ratio of tubelength sums for the tubes with and without pericyte contact. Error barsare reported as +SEM of two wells per group. * indicates p<0.05 comparedto DMSO group.

FIG. 5A shows the real-time fluorescent imaging of XPA-1-RFP pancreatictumor xenografts in the pancreas of Nestin-GFP mice (n=5/group) treatedwith either vehicle or compound 6 (50 mg/kg, ip, bid). Drug treatmentswere started 3 days after surgical orthotopic implantation (SOI) ofXPA1-RFP tumors and tumor progression was monitored every 3 days bywhole animal imaging. Scale bar=10 mm.

FIG. 5B shows the plot of tumor surface area over time for the vehicleand compound 6 treated groups. Tumor surface area was calculated byadding the total pixels of both the ventral and lateral images of thetumor. *p=0.034.

FIG. 5C shows the average body weight of the mice measured each day ofwhole animal imaging described in FIGS. 5A and 5B.

FIG. 5D illustrates the total weights of resected primary tumors on day15 post (SOI), *p=0.022.

FIG. 5E shows the representative fluorescent images of endothelial GFPexpression (GFP expression driven by the Nestin promoter) within theXPA1-RFP tumors after resection. Images taken 15 days post SOI. Scalebar=200 m.

FIG. 5F shows the plot of tumor vessel density from images acquired asin FIG. 5E. Blood vessels imaged as in FIG. 5E were converted to length(mm) and normalized to the tumor volume (mm³), *p=0.01.

FIG. 6A shows the sequence alignment of EGFR, B-RAF, and PDGFRβ primarysequences. 1uwh represents the co-crystal structure of sorafenib withBRAF and 1×kk is the co-crystal structure of GW 572016 and EGFR. Thesequence identity of the PDGFRβ and BRAF kinase domains (if the insertSEQ ID NO.1: LQHHSDKRRPPSAELYSNALPVGLPLPSHVSLTGESDGGYMDMSKDESVDYVPMLDMKGDVKYADIESSNYMAPYDNYVPSAPERTCRATLINESPVL in PDGFRβ is excluded) is29.1%, and overall similarity is 47.3% (opening gap penalty 15,extension gap penalty 1, using the program EMBOSS Align of EBI). Figurediscloses SEQ ID NOS 3-5, respectively, in order of appearance.

FIG. 6B illustrates the docking of compound 6 into PDGFRβ with an EGFRactivation loop. Because of the absence of sufficient crystallographicinformation for the activation loop in PDGFRβ, it can be modeled with avariety of possible loop conformations. By modeling the conformation ofthe activation loop in PDGFRβ near Gly857 using EGFR kinase (pdb id1×kk) as a template, we observe that Gly857 will be located in closeproximity to the phenyl ring of 6. Further addition of any atoms in thepara position would be sterically hindered by the loop. This would bepredicted to create an unstable position of 6 in the protein andpotentially explains why 4-Cl is not tolerated for PDGFRβ activity.

FIG. 7A shows a Western analysis comparing compound 3 and compound 6 (10μM) inhibition of bFGF or VEGF (50 ng/ml, 5 min stimulation) inducedphosphorylation of ERK in serum starved HUVECs as described herein. ERK2staining is shown for the loading control.

FIG. 7B shows the 1205Lu Melanoma cells which endogenously expressconstitutively active B-RAF V600E. The cells were starved overnight andtreated for 1 h with compound 6 (10 μM) to determine inhibition of theoncogenic B-RAF activity as measured by phosphorylation of MEK and ERK.

FIG. 7C shows the phosphorylation of C-RAF within the activation segmentat S338 or at the 14-3-3 binding site, S259, was determined by Westernanalysis. Endothelial cells were serum starved overnight and pretreatedwith compound 6 for 1 h before stimulation with either bFGF or VEGF at50 ng/ml for 5 min.

FIG. 7D shows the immunoprecipitation and Western analysis ofB-RAF/C-RAF heterodimerization. HUVECs were serum starved overnight andtreated with compounds as described in FIG. 7B. After growth factorstimulation with bFGF (F) or VEGF (V), C-RAF was immunoprecipitated(agarose conjugated C-RAF Ab, Santa Cruz) from 400 μg of protein fromtotal cell lysates and the presence of B-RAF (Santa Cruz) in theimmunoprecipitate was determined by Western blotting. Correspondingphosphorylation of ERK in the whole cell lysates (WCL) is shown in thebottom panel. Total ERK2 and total C-RAF were included in the blots asloading controls.

FIG. 7E shows the human umbilical vein endothelial cells (HUVECs) orhuman vascular smooth muscle cells (VSMC) were pre-treated with DMSO,compound 6, or sorafenib for 1 h followed by 5 minute stimulation withgrowth factor and lysis in RIPA buffer. 500 μg of protein was incubatedwith 3 μg of antibody (sc-432 for PDGFRβ and sc-6251 Flk-1/KDR, bothfrom Santa Cruz; and #05-149 from Upstate for FGFR1) for 1 h at 4° C.before the addition of Protein A/G PLUS agarose beads (Santa Cruz). Tomeasure autophosphorylation, immunoblots were carried out with aphospho-tyrosine antibody (PY-20, Santa Cruz) for one hour and detectedwith chemilluminescence. Membranes were stripped and reprobed for totalreceptor levels of PDGFRβ, Flk-1/KDR, and FGFR1, respectively (sc-339,sc-504, and 05-149, from Santa Cruz and Upstate, respectively).

FIG. 8A shows the labeling of the Tg:fli1-egfp zebrafish vasculature andquantification of the drug treatment effect on the ISV volume. Imageswere taken from FIG. 3A with major vascular structures labeled. In thehead: MCeV, middle cerebral vein; MsV, mesencephalic vein; PHS, primaryhead sinus; IOC, inner optic circle; PrA, prosencephalic artery. In thetail: DLAV, dorsal longitudinal anastomotic vessel; DA, dorsal aorta;PCV, posterior cardinal vein; ISV, intersegmental vessel; VTA, vertebralartery.

FIG. 8B shows a representative views of zebrafish embryos treated withcompound 6 and SU5416 as in FIG. 3A (images represent merged phase andGFP fluorescence views of the head and trunk regions of Tg:fli1-EGFPembryos). Scale bar=200 μm.

FIG. 8C shows the quantification of ISV volume from embryos treated asin FIG. 3A. To measure the intersegmental vessel (ISV) volume,individual ISVs were digitally isolated using the Imariscountersurface/isosurface functions. 30 independent ISVs from 4independent embryos were used for the measurement. Reported +/−sem.

FIG. 8D shows the quantification of ISV volume from embryos treated asin FIG. 3B. To measure the intersegmental vessel (ISV) volume,individual ISVs were digitally isolated using the Imariscountersurface/isosurface functions. 30 independent ISVs from 4independent embryos were used for the measurement. Reported +/−sem.

FIG. 9A shows that compound 6 inhibits bFGF induced angiogenesis in themouse Matrigel model. Mice (n=5) were injected in the flank withgrowth-factor depleted Matrigel loaded with 200 ng bFGF and treated for5 days with 50 mg/kg compound 6 via ip administration twice daily (bid).On day 5 after implantation of the Matrigel, the mice were administeredFITC-lectin to label blood vessels. The plugs were removed andvisualized by confocal microscopy followed by homogenization andquantification of total FITC. Error bars represent ±SEM. Scale bars=200m.

FIG. 9B shows that compound 6 inhibits phosphorylation of ERK in bloodvessels. Thin sections (5 μm) of the Matrigel plugs were immunostainedfor vessels with an EC mix (anti-Flk, anti-CD31, anti-VE-Cadherin, andanti-endoglin). Matrigel sections were immunostained for phospho-ERK todetect in vivo inhibitory activity of compound 6 by confocal microscopy.n=4 plugs/group. Scale bars=100 m.

FIG. 9C shows that compound 6 inhibits phosphorylation of PDGFRβ Y751 instromal cells. Thin sections (5 μm) of the Matrigel plugs wereimmunostained for pericytes and fibroblast with α-smooth muscle actinand for phospho-PDGFRβ Y751 levels. ECs were immunostained withanti-CD31. n=4 plugs/group. Scale bars=200 m.

FIG. 9D shows that compound 6 induces apoptosis in the endothelium butnot surrounding stromal cells in the Matrigel plugs. Thin sections (5μm) of the Matrigel plugs were immunostained for vessels with an EC mix(described as in 9B). TOPRO-3 was used as a nuclear counterstain andTUNEL staining was used to detect apoptotic nuclei. Images were takenusing confocal laser scanning microscopy. (n=4 plugs/group in 9B, 9C,9D). Scale bars=200 μm (9A, 9C, 9D);100 μm for (9B). n=4 plugs/group.Scale bars=200 μm.

FIG. 10A shows that compound 6 is orally active against orthotopic renalcell carcinoma. Real-time fluorescent imaging of SN12C-RFP renal cellsorthotopically injected into the flanks of nu/nu mice. Mice were treatedwith either vehicle or compound 6 (100 mg/kg, po, qd). Drug treatmentswere started 7 days after orthotopic injection and tumor growth wasvisualized every 7 days by whole animal imaging. The images shown are of4 mice in the vehicle and drug treated groups on day 26. Scale bar=10mm.

FIG. 10B shows that at the end of the study illustrated in FIG. 10A,both the left tumor bearing kidney and right kidney were extracted andweighed. The graph shows the mean difference in weight between thetumor-bearing kidney and the normal kidney in each animal. n=6animals/group. *p=0.05.

FIG. 11A illustrate that compound 6 is orally active against neointimalhyperplasia. Mouse carotid arteries were wire-injured as described inthe Methods. Animals (n=4/group) were treated the following day for 14days with either vehicle or compound 6 (100 mg/kg, po, qd). The carotidartery was excised on day 14, sectioned, and visualized with hematoxylinand eosin staining.

FIG. 11B shows the intimal/medial ratios measured for mice treated withvehicle and compound 6, *p<0.001.

FIG. 11C shows the corresponding percent stenosis (reflecting luminalnarrowing) calculated as described herein, *p<0.01. Error bars arereported as +SEM.

FIG. 11D shows the PDGFRβ autophosphorylation in the injured arteriesmeasured on day 14, 4 h post-dose.

FIG. 12A shows results of compound 6 as a potent inhibitor ofLymphangiogenesis. Images of local lymph node lymphangiogenesis modelbetween saline and VEGF-C matrigel.

FIG. 12B illustrates that compound 6 showed inhibition oflymphangiogenesis.

FIG. 13A shows the growth inhibitory properties of exemplary compoundson tumor cells. Compound 6 was profiled in the NCI60 panel by the NCIDevelopmental Therapeutics Program and demonstrated an average growthinhibitory concentration (GI50) of 490 nM with potent growth inhibitionacross the entire panel of cell lines.

FIG. 13B shows the cell proliferation curves of compounds 6, 35, or 37.

FIG. 13C shows the cell proliferation curves of sorafenib, PLX 4720 orL779,450.

FIG. 14 shows that sorafenib induces an increase in phosphorylation onCRAF S338, while Compound 6 does not promote this increase.

FIG. 15A shows that exemplary compounds cause a G2/M arrest atprometaphase. The microscopic analysis of tumor cells exposed tocompound 6, compound 3, PD0325901, paclitaxel, and sorafenib is shown.The microscopic analysis revealed that the cells exposed to compound 6appeared rounded and arrested in mitosis, whereas cells treated withsorafenib maintained their adhesive properties with evidence of intactmitotic function.

FIG. 15B shows brightfield images of XPA-1 cells treated for 20 hourswith PD0325901, compound 3, sorafenib, or compound 6 at 5 μM orpaclitaxel at 200 nM. Compound 3, sorafenib, and the MEK inhibitor(PD0325901) do not appear rounded and the cells appear similar to DMSOtreated control. Scale bar, 20 μm.

FIG. 16 shows exemplary compound 6 arresting a wide variety of tumorcell lines in G2/M. The graph represents the % of cells arrested in G2/Mafter treatment with 5 μM compound 6 compared to 0.1% DMSO control. Thegraph depicts G2/M quantification of human colon (HCT-116), pancreatic(Mia-Paca2, FG, XPA-1, BXPC3), breast (MB-MDA-231) and brain (U251)cancer cell lines. Error bars represent s.d. (n=4).

FIG. 17 demonstrates that compound 6 suppresses melanoma tumor growth invivo.

FIG. 18A demonstrates that compound 6 inhibits phosphorylation of S338in breast cancer, which correlates with breast cancer tumor growthsuppression. The MDA-MB-231 tumors were treated for three consecutivedays, and the tumors were resected 1 h following the final vehicle orcompound 6 dose and stained for phosphorylation on CRAF S338.

FIG. 18B displays the tumor growth profile after treatment with compound6 or vehicle.

FIG. 18C shows the pS338 CRAF levels in the MDA-MB231 cells in vitro.The cells were treated with DMSO (Ctrl), compound 6, or sorafenib at 5μM for 6 h and cell lysates were resolved on 10% SDS-PAGE andimmunoblotting was performed with the following antibodies: pS338 CRAF(Cell Signaling), CRAF (BD Pharmingen), and Actin (Sigma), all diluted1:1000.

FIG. 19A shows the potency of compounds 6, 31, 33, 36, and 37 in cellviability assays against tumor cells A549.

FIG. 19B shows the potency of compounds 6, 31, 33, 36, and 37 in cellviability assays against tumor cells T47D.

FIG. 19C shows the potency of compounds 6 and 31-37 in cell viabilityassays against tumor cells MDA-MB231.

DETAILED DESCRIPTION OF THE INVENTION

RAF kinase is an important convergent point downstream of FGFR andVEGFR2 signaling in endothelial cells and plays a critical role inendothelial cell survival during angiogenesis. The stromal compartmentis a major contributor to angiogenesis and tumor growth. This includespericytes associated with the newly forming endothelium, which stabilizethe vasculature and promote vascularization. PDGFRβ is a receptortyrosine kinase (RTK) that is essential for promoting proper pericytefunction, which stabilizes blood vessels and enables vessel maturation.PDGFRβ signaling potentiates pericyte recruitment to newly formingvessels and the secretion of pro-angiogenic molecules such as VEGFA,FGF2, and Ang1 in the local microenvironment. This promotes vesselstabilization and remodeling of the immature vascular network to ahighly ordered network. Maintenance of the vascular compartment isdependent upon paracrine loops such as the secretion of PDGF-BB andFGF2, which lead to increased expression of FGFR1 on VSMCs and PDGFRα/βon ECs, respectively. Therefore, the homeostasis of the mural andvascular compartments is critical for efficient angiogenesis.

Thus inhibiting these two compartments simultaneously would initiate apotent inhibition of angiogenesis. While broad-spectrum receptortyrosine kinase inhibitors are available, the goal was to designcompounds with a narrow kinase profile to selectively inhibit relevantpathways involved in neovascularization. RAF kinase was targeted sincethis kinase is a downstream of multiple RTKs and is required for ECproliferation and survival, and PDGFRβ is a critical for pericyterecruitment and vessel maturation.

Due to the hydrophobic interactions and specific hydrogen bondingrequired for type II inhibition, the allosteric site adjacent to thekinase active site may be utilized to improve specificity over the typeI Inhibitors that interact solely with the active kinase conformation inthe highly conserved hinge region. Imatinib (1) and sorafenib (2) wereco-crystallized with their respective targets, B-RAF and Abl kinasedomains, and shown to interact in part with the allosteric site in the“DFG-out” conformation—referred to as “type II” inhibition. Based on thebinding mode of imatinib (1) and sorafenib (2), compounds of anamino-triazole scaffold designed to target the allosteric site of bothPDGFRβ and B-RAF using a combination of in silico screening and in vitrobioassays were prepared.

1

2

Compound R₁ R₂ R₃ Z 3 H NH₂ H NH 4 NH₂ NH₂ H NH 5 NH₂ NH₂ Cl NH 6 MeSNH₂ H NH 7 MeS H H NH 8 MeS NH₂ Cl NH 9 NH₂ MeO H NH 10 NH₂ NH₂ H O 11MeS NH₂ H O 13 MeS H Cl NH 14 MeS(O)₂ NH₂ H NH 15 SEt NH₂ H NH 16S-^(tert-)Bu NH₂ H NH 17 NH₂ S-^(tert-)Bu Cl NH 18 MeS H Br O 19 NH₂ SMeF NH 20 NH₂ SEt I NH 21 Et NH₂ Cl NH 22 CF₃S H Cl NH 23 NH₂ CF₃S H NH 24CF₃S NH2 H NH 25 CF₃S NH2 Cl NH 26 CF₃S H H NH 27 NH₂ MeO Cl NH 28 H NH₂Cl NH 29 NH₂ H H NH 30 MeO NH2 H NH

Compound R₁ R₂ Ar Z′ 31 SMe NH₂

N 32 EtO NH₂

N 33 MeS NH₂

N 34 MeS NH₂

N 35 MeS NH₂

N 36 MeS NH2

N 37 MeS NH₂

C

Among them, compound 6, would have been disregarded using traditional invitro ATP-dependent kinase assays; at 10 μM, compound 6 did not inhibitany of its targets in this screening format (data not shown). ForPDGFRβ, the cellular IC50 and biochemical Kd matched quite well as bothwere approximately 500 nM (FIG. 1B and Table 3), demonstrating asignificant difference between the activity of compound 6 in cell-basedvs. activated kinase assays. This is not surprising since therecombinant enzymes are not subject to the same conformationalinactivation as the intact cell-associated enzymes. Although compound 6is predicted to stabilize the inactive conformation of PDGFRβ or B-RAF,it might not be expected to suppress the activity of a recombinantactivated form of this enzyme in vitro that is not subject to negativeregulation. Similarly, imatinib, is 200-fold more active against the Ablkinase domain when the activation loop is unphosphorylated.

Among these compounds, compound 6 does not inhibit activated kinases invitro and possesses dual inhibition of PDGFRβ/RAF activity that producesa potent anti-angiogenic effect which is not seen with the inhibition ofeither target alone. Combination of separate inhibitors of PDGFRβ andRAF reproduces the anti-angiogenic effects of compound 6 in both thezebrafish in vivo and a pericyte/endothelial cell tube formation assayin vitro, and further validates the dual targeting of PDGFRβ and RAF asa synergistic approach. Additionally, compound 6 suppresses tumor growthin orthotopic tumor models of pancreatic and renal cell carcinoma. Whilecompound 6 inhibited cellular PDGFR and RAF, it also disrupted Flt3 andKIT (Table 3). However, Flt3 and KIT are not essential to the biologicalactivity of compound 6 since compound 3, which blocks Flt3, Kit, andPDGFR but does not inhibit RAF, failed to disrupt vessel formation (FIG.3A-B). This result further supports the synergy of inhibiting both PDGFRand RAF for increased anti-angiogenic activity as observed in FIG. 4using completely different chemotypes.

Similarly, the allosteric site adjacent to the kinase active site inpseudokinases (e.g., HER3, EphB6, CCK4, KSR, Trb3, GCN2, TRRAP, LIK andCASK), proteins having a kinase-like domain, may be utilized to modulatediverse cellular processes of the pseudokinases.

In accordance with the present invention, the development of selectiveinhibitors directed against PDGFRβ and RAF (e.g. A-RAF, B-RAF andC-RAF), and define a synergistic combination which leads to effectiveinhibition of angiogenesis and tumor growth are discovered. Although newclinically approved kinase inhibitors work through broad-spectruminhibition and opportune target combinations, invention methods areprovided to narrow the kinase profile to selective combinations whichprovide great synergy. This approach will ultimately increase thetherapeutic window of these agents and improve the chance of providingtherapeutic efficacy with minimal side effects.

Process of Preparing Exemplary Compounds

The exemplary compounds described herein, in some instances, areprepared according to the known procedures or the novlel proceduresdisclosed herein.

In some embodiments, there are provided processes for preparing atriazole compound of formula I:

comprising reacting a compound of formula II,

with a compound of formula (III),

in the presence of a base, wherein W is selected from the groupconsisting of pyridine, pyridazine, pyrimidine, pyrazine, triazine,quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline,1,2,4-benzotriazine and azaindole; X is O, S or NH; R₁ and R₂ areindependently a hydrogen, optional substituted alkyl, halogen, optionalsubstituted amine, —NH₂, —OH, optional substituted alkoxy, optionalsubstituted thioalkyl, —SCF₃, optional substituted alkylsulfinyl or anoptional substituted alkylsulfonyl; R₃ is independently a hydrogen,halogen substituted C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkoxy, or,optionally, two R₃ are joined to form a 5 to 7 membered carbocycle; andm is 1-5. (See scheme 1)

In one embodiment, the triazole formation is achieved with good yieldunder suitable solvent with a base, preferable an amine base; morepreferable a 2,6-lutidine. In certain embodiments, the molar ratio ofthe compound of formula II to the compound of formula III is in therange from 0.6:1 to 0.9:1.

The triazole formation provided herein is applicable to many hydrazideprecursors. For example, the reaction is applicable where W is pyridine,pyridazine, pyrimidine, pyrazine, triazine, quinoline, isoquinoline,cinnoline, quinazoline, quinoxaline, 1,2,4-benzotriazine, azaindole, orthe like. The linkage between W and the phenyl ring can be an ether,thioether or amine linkage. The S-methylthiourea of formula (III) isprepared in situ from its thiourea precursor upon methylation (e.g.MeI). When suitable solvent (determined by the solubility of thehydrazide precursors and the in situ S-methylthiourea) and base areused, good yield with satisfactory purity can be achieved. The triazolecompounds can then go to next step directly (e.g. salts preparation)without time-consuming and/or labor-intensive column chromatography.

For example, without limitation, a skilled artisan may use the followinghydrazide precursors.

In some embodiments, a compound of formula I, preferably of formula Ia,

can be prepared from reacting a compound of formula IIa,

with a compound of formula (IIIa),

in the presence of a base (Scheme 2), wherein R₁ and R₂ areindependently a hydrogen, optional substituted alkyl, halogen, optionalsubstituted amine, —NH₂, —OH, optional substituted alkoxy, optionalsubstituted thioalkyl, —SCF₃, optional substituted alkylsulfinyl or anoptional substituted alkylsulfonyl and R₃ is a hydrogen, halogensubstituted C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkoxy. In certainembodiments, the molar ratio of the compound of formula IIa to thecompound of formula IIIa is in the range from 0.6:1 to 0.9:1. Theprocesses have been optimized to provide compounds of formula (Ia) withsatisfactory purity and thus require little or no chromatographicpurification. In general, after the processes, the crude is ready fornext step, e.g., to prepare its salts.

In some embodiments, a compound of formula I, preferably of formula Ib,

can be prepared from reacting a compound of formula IIa,

with a compound of formula (IIIb),

in the presence of a base, wherein R₁ and R₂ are independently ahydrogen, optional substituted alkyl, halogen, optional substitutedamine, —NH₂, —OH, optional substituted alkoxy, optional substitutedthioalkyl, —SCF₃, optional substituted alkylsulfinyl or an optionalsubstituted alkylsulfonyl; R^(m) is C₁₋₆ alkyl, or halogen substitutedC₁₋₆ alkyl; R₃ is independently a hydrogen, C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, orC₃₋₁₀cycloalkyl; or, optionally, R^(m) and R₃ are joined to form a fiveto seven membered carbocycle; and n is 0-4. In certain embodiments, themolar ratio of the compound of formula IIa to the compound of formulaIIIb is in the range from 0.6:1 to 0.9:1. The processes have beenoptimized to provide compounds of formula (Ib) with satisfactory purityand thus require little or no chromatographic purification. In general,after the processes, the crude is ready for next step, e.g., to prepareits salts.

Nature of the base was found to be important to maximize the yield. Insome embodiments, the base used for the processes is an amine base,preferably a 2,6-lutidine.

It is also determined that the choice of solvent would affect the purityand the yield of the triazole formation. In some embodiments, thesolvent used for the processes of triazole formation herein isacetonitrile, DMF, N-methyl-pyrrolid-2-one (NMP), ^(t)BuOH or ^(i)PrOH.

In other embodiments, there are provided processes of preparingcompounds of formula (I), (Ia) or (Ib) wherein R₁ is a hydrogen, —NH₂,optional substituted alkoxy, optional substituted thioalkyl, —SCF₃,optional substituted alkylsulfinyl or an optional substitutedalkylsulfonyl. For example, R₁ may be —H, —NH₂, —OMe, —OEt, —SMe, —SEt,—S^(t)Bu, —SO₂Me, —SCF₃, or the like.

In other embodiments, there are provided processes of preparingcompounds of formula (I), (Ia) or (Ib) wherein R₂ is a hydrogen, NH₂, oran optional substituted alkoxy. For example, R₂ may be —H, —NH₂, —NHMe,—OMe, —OEt, —OCF₃, or the like.

In other embodiments, there are provided processes of preparingcompounds of formula (I), (Ia) or (Ib) wherein R₁ and R₂ areindependently an optional substituted amine. For example, R₁ and/or R₂may be —NH₂, —NHMe, or the like.

In other embodiments, there are provided processes of preparingcompounds of formula (I), (Ia) or (Ib) wherein R₁ is an optionalsubstituted thioalkyl and R₂ is an optional substituted amine. Forexample, R₁ may be —SMe, —SEt, —S^(t)Bu, —SCF₃, or the like and R₂ maybe —NH₂, —NHMe, or the like. In certain embodiments, R₁ is a MeS and R₂is a NH₂.

In the case of 1,2,4-triazoles, there exist three tautomeric structures,as shown below:

Which tautomeric structure is prevailing depends on the substituents onthe triazole moiety and on the reaction conditions. As known to thosehaving ordinary skill in the art, typically, 1H-1,2,4-triazole is themost common tautomeric form, especially if an amino substituent isattached to the ring. Even though all three tautomeric structures can bepresent, all the generic structures and all the examples having1,2,4-triazole moiety are shown herein in one tautomeric form, such as4H-1,2,4-triazole, for simplicity and for the comparison with its directanalogues, such as examples containing 1,3,4-oxadiazole moiety. Usingonly 4H-tautomeric form to draw the structures for the sake ofsimplicity, does not imply that the triazole compounds provided hereinexist in that particular tautomeric form.

In according with the present invention, there are provided processesfor preparing a compound of formula IV:

comprising reacting a compound of formula V,

with a compound of formula (VI),

in the presence of a base wherein L is a leaving group e.g., a chloride,tosylate or other suitable leaving group; Y is —SH, —OH, or —NH₂; R₁ andR₂ are independently hydrogen, optional substituted alkyl, halogen,optional substituted protected amine, optional substituted alkoxy,optional substituted thioalkyl, CF₃S, optional substituted alkylsulfinylor optional substituted alkylsulfonyl and R₄ is a C₁₋₆ alkyl. A compoundof formula IV is then converted to a hydrazide suitable for triazoleformation (Scheme 3).

In certain embodiments, the compound of formula IV is a compound havingthe structure of formula IVa:

and the compound of formula V is a compound having the structure of Va,

and the compound of formula VI is an alkyl hydroxybenzoate. Theprocesses for preparing a compound of formula IV provided herein areunder very mild conditions, e.g. at about 25-35° C. (room temperature)when R₁ and/or R₂ are a Boc protected amine. The processes have beenoptimized to provide compounds of formula IV or IVa with satisfactorypurity and thus require little or no chromatographic purification. Ingeneral, after the processes, the crude can be used for next step.

In some embodiments, the base used for the processes of preparingcompounds of formula IV or IVa is an amine base; preferably a1,4-diazabicyclo[2.2.2]octane. In other embodiments, methyl4-hydroxybenzoate is used in the processes.

In other embodiments, there are provided processes of preparingcompounds of formula IV or IVa wherein R₁ is a hydrogen, optionalsubstituted protected amine, optional substituted alkoxy, optionalsubstituted thioalkyl, CF₃S, optional substituted alkylsulfinyl or anoptional substituted alkylsulfonyl. For example, R₁ may be —H, —NH₂,—OMe, —OEt, —SMe, —SEt, —StBu, —SO₂Me, —SCF₃, or the like.

In other embodiments, there are provided processes of preparingcompounds of formula IV or IVa wherein R₂ is a hydrogen, optionalsubstituted protected amine, or an optional substituted alkoxy. Forexample, R₂ may be —H, —NH₂, —NHMe, —OMe, —OEt, —OCF₃, or the like.

In other embodiments, there are provided processes of preparingcompounds of formula IV or IVa wherein R₁ and R₂ are independently anoptional substituted protected amine; preferably the optionalsubstituted protected amine is protected by an acid labile protectinggroup such as Boc. For example, R₁ and/or R₂ may be —NH₂, —N(Boc)Me,—N(Boc)₂, or the like. Preferably, R₁ and R₂ are independently a—N(Boc)₂.

In other embodiments, there are provided processes wherein R₁ is anoptional substituted thioalkyl and R₂ is an optional substitutedprotected amine, preferably an amine with an acid labile protectinggroup such as -Boc. For example, R₁ may be —SMe, —SEt, —S^(t)Bu, —SCF₃,or the like and R₂ may be —NH(Boc), —N(Boc)Me, N(Boc)₂, or the like. Insome embodiments, there are provided processes of preparing compounds offormula IV or IVa wherein R₁ is a —SMe and R₂ is a —N(Boc)₂.

Examples of Methods of Dosing and Treatment Regimens

In one aspect, the compositions containing the compounds (i.e.,allosteric kinase inhibitors described herein) are administered forprophylactic and/or therapeutic treatments. In therapeutic applications,the compositions are administered to a patient already suffering from adisease, disorder, or condition, in an amount sufficient to cure or atleast partially arrest the symptoms of the disease, disorder, orcondition. Amounts effective for this use will depend on the severityand course of the disease, disorder, or condition, previous therapy, thepatient's health status, weight, and response to the drugs, and thejudgment of the treating physician.

In prophylactic applications, compositions containing the compounds(i.e., allosteric kinase inhibitors described herein) are administeredto a patient susceptible to or otherwise at risk of a particulardisease, disorder or condition. Such an amount is defined to be a“prophylactically effective amount or dose.” In this use, the preciseamounts also depend on the patient's state of health, weight, and thelike. In some embodiments, when used in a patient, effective amounts forthis use depend on the severity and course of the disease, disorder orcondition, previous therapy, the patient's health status and response tothe drugs, and the judgment of the treating physician.

In some embodiments, the case wherein the patient's condition does notimprove, upon the doctor's discretion the administration of thecompounds (i.e., allosteric kinase inhibitors described herein) areadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease, disorder, or condition.

In some embodiments, wherein the patient's status does improve, upon thedoctor's discretion the administration of the compounds (i.e.,allosteric kinase inhibitors described herein) are given continuously;alternatively, the dose of drug being administered is temporarilyreduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”). In other embodiments, the length of the drug holidayvaries between 2 days and 1 year, including by way of example only,about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 10 days, about 12 days, about 15 days, about 20days, about 28 days, about 35 days, about 50 days, about 70 days, about100 days, about 120 days, about 150 days, about 180 days, about 200days, about 250 days, about 280 days, about 300 days, about 320 days,about 350 days, or about 365 days. In further embodiments, the dosereduction during a drug holiday is from about 10% to about 100%,including, by way of example only, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or about 100%.

Once improvement of the patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, in other embodiments,the dosage or the frequency of administration, or both, are reduced, asa function of the symptoms, to a level at which the improved disease,disorder or condition is retained. In further embodiments, patientswill, however, require intermittent treatment on a long-term basis uponany recurrence of symptoms.

In other embodiments, the amount of a given agent that corresponds tosuch an amount varies depending upon factors such as the particularcompound, disease, disorder, or condition and its severity, the identity(e.g., weight) of the subject or host in need of treatment, butnevertheless is routinely determined in a manner according to theparticular circumstances surrounding the case, including, e.g., thespecific agent being administered, the route of administration, thecondition being treated, and the subject or host being treated. In someembodiments, however, doses employed for adult human treatment aretypically in the range of about 0.02 to about 5000 mg per day or about 1to about 1500 mg per day. In further embodiments, the desired dose isconveniently presented in a single dose or as divided doses administeredsimultaneously (or over a short period of time) or at appropriateintervals, for example as two, three, four or more sub-doses per day.

In some embodiments, the pharmaceutical composition described herein arein unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of one or more compound. in otherembodiments, the unit dosage is in the form of a package containingdiscrete quantities of the formulation. Non-limiting examples arepackaged tablets or capsules, and powders in vials or ampoules. Inanother embodiment, aqueous suspension compositions are packaged insingle-dose non-reclosable containers. In further embodiments,multiple-dose reclosable containers are used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection are presented in unit dosageform, which include, but are not limited to ampoules, or in multi-dosecontainers, with an added preservative.

The daily dosages appropriate for the compounds (i.e., allosteric kinaseinhibitors described herein) described herein described herein are fromabout 0.01 to about 2.5 mg/kg per body weight. An indicated daily dosagein the larger mammal, including, but not limited to, humans, is in therange from about 0.5 mg to about 100 mg, conveniently administered individed doses, including, but not limited to, up to four times a day orin extended release form. Suitable unit dosage forms for oraladministration include from about 1 to about 50 mg active ingredient.The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. In furtherembodiments, such dosages are altered depending on a number ofvariables, not limited to the activity of the compound used, thedisease, disorder, or condition to be treated, the mode ofadministration, the requirements of the individual subject, the severityof the disease, disorder, or condition being treated, and the judgmentof the practitioner.

In yet further embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and in some embodiments is expressed asthe ratio between LD₅₀ and ED₅₀. In other embodiments, the data obtainedfrom cell culture assays and animal studies is used in formulating arange of dosage for use in human. In some embodiments, the dosage ofsuch compounds lies within a range of circulating concentrations thatinclude the ED₅₀ with minimal toxicity. In yet further embodiments, thedosage varies within this range depending upon the dosage form employedand the route of administration utilized.

Lymphangiogenesis, Angiogenesis and/or Growth of a Tumor

Provided herein are methods for inhibiting or preventinglymphangiogenesis, angiogenesis and/or growth of a tumor, whichcomprises contacting the tumor with a compound that (i) stabilizes aprotein kinase in the inactive state and (ii) is not an ATP competitiveinhibitor of the protein kinase in the active state. In someembodiments, the compound for the methods is a selective type IIinhibitor; preferably a selective type II inhibitor of RAF kinase (e.g.B-RAF kinase); more preferably the compound is also a selective type IIinhibitor of a PDGF receptor. In some embodiments, the compound for themethods modulates A-RAF. In other embodiments, the compound for themethods inhibits the heterodimerization of BRAF with CRAF. In someembodiments, the compound inhibits the phosphorylation of S338 of C-RAF.In another embodiment, the compound does not inhibit active kinasesselected from the group B-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, andPDGFRβ. In some embodiments, the compound is an allosteric inhibitor ofPDGFRα, PDGFRβ, Flt3 and/or c-Kit. In some embodiments, the compound hasthe structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

The term “alkyl” as used herein, means a straight, branched chain, orcyclic (in this case, it would also be known as “cycloalkyl”)hydrocarbon containing from 1-10 carbon atoms. Illustrative examples ofalkyl include, but are not limited to, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylhexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

The term “carbocyclic” or “carbocycle” refers to a ring wherein each ofthe atoms forming the ring is a carbon atom. Carbocycle includes aryland cycloalkyl. The term thus distinguishes carbocycle from heterocycle(“heterocyclic”) in which the ring backbone contains at least one atomwhich is different from carbon (i.e a heteroatom). Heterocycle includesheteroaryl and heterocycloalkyl. Carbocycles and heterocycles can beoptionally substituted. The following illustrates a few exemplarycarbocycles

The term “optionally substituted” as defined herein, means thereferenced group is substituted with zero, one or more substituents asdefined herein.

As used herein, the term “sulfinyl” refers to a —S(═O)—R, where R isselected from the group consisting of alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heterocycloalkyl (bondedthrough a ring carbon).

As used herein, the term “sulfonyl” refers to a —S(═O)₂—R, where R isselected from the group consisting of alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heterocycloalkyl (bondedthrough a ring carbon).

The lymphatic vascular system plays an important role in the maintenanceof tissue fluid homeostasis, in the afferent phase of the immuneresponse, and in the metastatic spread of cancers. Vascular endothelialgrowth factor-C (VEGF-C) and VEGF-D are secreted glycoproteins thatactivate VEGF receptor-2 (VEGFR-2) and VEGFR-3 (Achen et al., (1998)Proc. Natl. Acad. Sci. USA 95: 548-553; Joukov et al., (1996) EMBO J.15: 290-298)—cell surface receptor tyrosine kinases that are expressedpredominantly on blood vascular and lymphatic endothelia respectively(for review see Stacker et al., FASEB J. 16: 922-934, 2002). VEGFR-3signals for lymphangiogenesis (Veikkola, et al., (2001) EMBO J. 20:1223-1231) whereas VEGFR-2 is thought to signal for angiogenesis. HumanVEGF-C and VEGF-D stimulate both angiogenesis and lymphangiogenesis invivo (See e.g. Byzova et al., (2002) Blood 99: 4434-4442; Veikkola etal., (2001) EMBO J. 20: 1223-1231; Marconcini et al., (1999) Proc. Natl.Acad. Sci. USA 96: 9671-9676).

There is increasing evidence that lymphatic vessels also activelyparticipate in acute and chronic inflammation. Lymphangiogenesis hasalso been observed in experimental models of chronic airwayinflammation. UVB irradiation of the skin also results in enhancedexpression of vascular endothelial growth factor (VEGF)-A, and systemicblockade of VEGF-A led to diminished UVB-induced lymphatic vesselabnormalities and skin inflammation in mice, indicating thatVEGF-A-mediated impairment of lymphatic vessel function promotes edemaformation and inflammation. Accordingly, selective type II inhibitor ofprotein kinase (e.g. B-RAF kinase) stabilizing a protein kinase in theinactive state with a compound that is not ATP competitive inhibitors ofthe protein kinase in the active state, can be used to block theactivation of VEGF-C and VEGF-D and thereby inhibit angiogenesis,lymphangiogenesis and other biological effects induced by partiallyprocessed or fully processed VEGF-C or VEGF.

Pseudokinase

In some embodiments, the methods described here for treating cancer in ahuman subject comprising administering to a patient in need a compoundthat binds a pseudokinase. In certain embodiments, the pseudokinase is akinase suppressor of Ras (KSR).

Several structural and sequence homology studies of protein kinasedomains have revealed a consensus of what are the common motifs that arerequired for catalytic activity. In some instances, these compriseresidues that are required for nucleotide (ATP) binding, metal ion(Mg2+) binding and residues required for phosphoryl group transfer.There are 518 known human protein kinases, representing the third mostcommon functional domain. Interestingly, about 10% of the kinome appearto lack at least one of the motifs required for catalysis and have beentermed pseudokinases.

Several studies show that mutations affecting pseudokinase domainsunderlie the dysregulation of catalytic activity of severalclinically-important kinases, including LKB1, Raf and Jak2, by theirpartner pseudokinase regulators, STRAD, KSR and the Jak2 JH2 domain,respectively. These studies provide a link between pseudokiase-mediateddysregulation of signal transduction and a number of diseases includingcancers and blood cell malignancies. Exemplary pseudokinases areprovided herein.

STRADα

The Ste20 related adaptor (STRADα and STRADβ) isoforms are part of theLKB1 heterotrimeric tumour suppressor complex. Together with the adaptorprotein MO25, STRAD activates the LKB1 kinase through an allostericmechanism that does not require LKB1 activation loop phosphorylation.(Zeqiraj et al., Science 2009; 326(5960): 1707-1711) Despite the changesin the glycine-rich loop, (the third consensus glycine being replaced byMet83, the crystal structure of STRADα revealed that STRADα is capableof binding ATP with low nanomolar affinity, and retains a kinase foldthat that is typical of the canonical ‘active’ kinase conformation.(Zeqiraj et al., PLoS Biol 2009; 7(6):e1000126) The active conformationof STRADα was shown to be modulated by its binding partner MO25, as wellas ATP. Loss of ATP and MO25 binding impinges on the ability of STRAD toactivate the LKB1 kinase. This suggests that the ‘active’ conformationof STRADα plays a key role.

ILK

Integrin-linked kinase (ILK) is a 59 kDa protein originally identifiedwhile conducting a yeast-two hybrid screen with integrin 131 as the baitprotein (Hannigan et al., 1996). Since its discovery, ILK has beenassociated with multiple cellular functions including cell migration,cell proliferation, cell-adhesions, signal transduction. Integrin-linkedkinase (ILK), interacts with the cytoplasmic domain of beta-1 integrin.This gene encodes a serine/threonine protein kinase with 4 ankyrin-likerepeats, which associates with the cytoplasmic domain of beta integrinsand acts as a proximal receptor kinase regulating integrin-mediatedsignal transduction. Multiple alternatively spliced transcript variantsencoding the same protein have been found for this gene. ILK is part ofa heterotrimeric complex together with PINCH and parvin (the so-calledIPP complex). See e.g., Legate, et al., Nat Rev Mol Cell Biol 2006;7(1):20-31. A recent crystal structure of ILK bound to α-Parvin hasuncovered the molecular basis of ILK function and explains why ILK isincapable of phosphorylating any substrates (Fukuda et al., Mol Cell2009; 36(5):819-830). A striking feature of the ILK-α-parvin complexstructure is the presence of ATP in the ILK nucleotide binding pocket,despite several non-conservative substitutions of crucial glycineresidues in the glycine-rich loop.

HER3

HER3/ErbB3 is a member of the human epidermal growth family (HER) oftyrosine kinase receptors that also includes HER1/ErbB1, HER2/ErbB2 andHER4/ErbB4. Of the four members, HER3 is classified as a pseudokinasebecause it lacks two of the eleven residues important for catalysis.Upon ligand binding to the EGF receptor, the intracellular kinasedomains undergo homodimerisation and heterodimerisation resulting in theformation of active asymmetric dimmers. ErbB3/Her3 is an enzyme that inhumans is encoded by the ERBB3 gene. This gene encodes a member of theepidermal growth factor receptor (EGFR) family of receptor tyrosinekinases. This membrane-bound protein has a neuregulin binding domain butnot an active kinase domain. It therefore can bind this ligand but notconvey the signal into the cell through protein phosphorylation.However, it does form heterodimers with other EGF receptor familymembers which do have kinase activity. Heterodimerization leads to theactivation of pathways which lead to cell proliferation ordifferentiation. Amplification of this gene and/or overexpression of itsprotein have been reported in numerous cancers, including prostate,bladder, and breast tumors.

VRK3

VRK3 (vaccinia related kinase 3) lacks catalytic activity owing to thesubstitution of six out of eleven active site residues. The structure ofVRK3 reveals how non-conservative substitutions of these catalyticmotifs compromise VRK3 catalytic competence. Of detrimental effect toATP binding and hence catalytic activity, are the substitution of asmall glycine residue from the glycine-rich loop (residue Asp175) andresidue Gln177. It is predicted that the substitution clashes with thephosphate moiety of ATP. Consistent with these structural observations,VRK3 is incapable of binding nucleotides. The VRK3 structure is similarto the structure of the closely related active kinase VRK2. Recentstudies suggest that VRK3 direct binding inhibits the vacciniaH1-related (VHR) phosphatase, a dual-specificity phosphatase thatdephosphorylates and inactivates ERK (See for example, Kang, et al., NatCell Biol 2006; 8(8):863-869; Kang, et al., Biochim Biophys Acta 2008;1783(1):49-58). Thus, VRK3 regulates MAP kinase signalling throughinhibition of ERK activity.

KSR

The kinase suppressor of Ras (KSR) family of proteins comprise aconserved group of molecular scaffolds that function as modulators ofRas signaling by bringing together the different components of theRaf/MEK/ERK cascade. These predicted pseudokinases act as scaffoldsbringing together the three components of the MAP kinase pathway(MAPKKK, MAPKK and MAPK), thus regulating signalling output andpotentiation. The pseudokinase domain of KSR1 binds MEK and RAF, whereasERK is recruited to the signalling complex via a conserved domainN-terminal to the pseudokinase domain. In addition, recent work thatestablished KSR2 as an important scaffold (similar to KSR1) of MAPkinase signalling, reveals KSR2 can be regulated by dephosphorylation bycalcineurin in response to changing Ca2+ levels. (Dougherty, et al., MolCell 2009; 34(6):652-662) KSR proteins are similar to members of the Raffamily of S/T kinases, and as such are included in the tyrosine-likekinase (TLK) group of kinases. All KSR proteins identified so far haveall or some of five distinct domains named Conserved Area (CA) 1 to CA5.

Treatment of Cancer

In some embodiments, the methods described here for treating cancer in ahuman subject comprise administering to a patient in need a compoundthat (i) stabilizes a protein kinase in the inactive state and (ii) isnot an ATP competitive inhibitor of the protein kinase in the activestate. In certain embodiments, the cancer is resistant, refractory ornon-responsive to a type I inhibitor of the protein kinase. In certainembodiments, the cancer is resistant, refractory or non-responsive to apan-RAF kinase drug or an ATP-competitive inhibitor. In certainembodiments, the cancer is resistant, refractory or non-responsive to adrug selected from Sorafenib, PLX4032, XL281, RAF265, 885-A, ZM336372,L-779450, AZ628, AAL881, LBT613, MCP110, 17-DMAG, CI1040,AZD6244/ARRY142886, PD0325901, SB590885, DP3346, and DP2514. In certainembodiment, the cancer is resistant, refractory or non-responsive to aVEGF-targeted therapy. In certain embodiments, the cancer is associatedwith a mutant form of RAF kinase; preferably the mutant form is a BRAFkinase selected from a mutant T529I, T529N, G464A, G464E, G464V, G466A,G466E, G466V, G469A, G469E, N581S, E586K, F595L, G596R, L597V, L597R,T599I, V600E, and K601E. In another embodiment, the mutant form is aCRAF gatekeeper mutant selected from T421N and T421I. In otherembodiments, the cancer is selected from melanoma, breast cancer, coloncancer, pancreatic cancer, lung cancer (e.g. non-small cell lungcancer), kidney cancer, glioblastoma, and colon cancer. In certainembodiments, the cancer is characterized by stroma rich tumors. Incertain embodiments, the cancer has a mutant or aberration selected fromN-Ras, B-RAF(V600E), B-RAF/Ras, HER1, K-Ras, or PI3K. In certainembodiments, the cancer exhibits up-regulation of the RAF-MEK-ERKpathway. In some embodiments, the compound for the methods is aselective type II inhibitor; preferably a selective type II inhibitor ofRAF kinase (e.g. B-RAF kinase); more preferably the compound is also aselective type II inhibitor of a PDGF receptor. In some embodiments, thecompound modulates A-RAF. In other embodiments, the compound for themethods inhibits the heterodimerization of BRAF with CRAF. In someembodiments, the compound inhibits the phosphorylation of S338 of C-RAF.In another embodiment, the compound does not inhibit active kinasesselected from the group B-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, andPDGFRβ. In some embodiments, the compound arrests tumor cells in G2/M.

In some embodiments, the compound is an allosteric inhibitor of PDGFRα,PDGFRβ, Flt3 and/or c-Kit. In some embodiments, the compound comprisesan amino-triazole scaffold. In some embodiments, the compound has thestructure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

In certain embodiments, the compound is administered orally to thepatient in need. In some embodiments, the patient is also provided witha therapy selected from anti-angiogenic therapy, chemotherapy orradiation therapy. In other embodiments, the response of the patient tothe compound is monitored by inhibition of the phosphorylation of S338of CRAF.

In some embodiments, cancers that are treated by the methods providedherein include, but are not limited to: Cardiac: sarcoma (angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamouscell, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor, chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiforme, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cord(neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia)!ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clearcell carcinoma, unclassified carcinoma], granulosa-thecal cell tumors,Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva(squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma,fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopiantubes (carcinoma); Hematoloqic: blood (myeloid leukemia [acute andchronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia,myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.

In some embodiments, acute myelocytic leukemia (AML) and/or acutelymphocytic leukemia (ALL) are treated using compounds (i.e., allosterickinase inhibitors described herein) in monotherapy or combinationtherapy.

ASK1-Mediated Apoptosis

In other embodiments, the methods described here for preventinginhibition of ASK1-mediated apoptosis in a cell comprise contacting thecell with a compound that (i) stabilizes the protein kinase in theinactive state and (ii) is not an ATP competitive inhibitor of theprotein kinase in the active state. In some embodiments, the compoundfor the methods is a selective type II inhibitor; preferably a selectivetype II inhibitor of RAF kinase (e.g. B-RAF kinase); more preferably thecompound is also a selective type II inhibitor of a PDGF receptor. Insome embodiments, the compound for the methods modulates A-RAF. In otherembodiments, the compound for the methods inhibits theheterodimerization of BRAF with CRAF. In some embodiments, the compoundinhibits the phosphorylation of S338 of C-RAF. In another embodiment,the compound does not inhibit active kinases selected from the groupB-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, and PDGFRβ. In someembodiments, the compound is an allosteric inhibitor of PDGFRα, PDGFRβ,Flt3 and/or c-Kit. In some embodiments, the compound described hereinarrests tumor cells in G2/M. For example, exemplary compound 6 arreststumor cells such as HCT-116, Mia-Paca2, FG, XPA-1, BXPC3, MDA-231 andU251 in G2/M. In some embodiments, the compound has the structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

Apoptosis or programmed cell death is a physiologic process that plays acentral role in normal development and tissue homeostasis. Many factorsinteract in complex pathways to lead to cell death or cell survival.Genotoxic stress induced by anticancer drugs can lead to apoptosis ofboth angiogenic endothelia cells and proliferating tumor cells. However,growth factors such as basic fibrolast growth factor (bFGF) and VEGFpresent within the tumor microenvironment by suppressing apoptoticmechanisms in these cells. It was identified that apoptosissignal-regulating kinase 1 (ASK1) can be a target of bFGF-mediatedsurvival signaling in ECs (Alavi, et al. (2007) Cancer Res 67:(6) 2766).Specifically, it is thought that bFGF stimulation promotes the formationof a Raf-1/ASK1 complex at the mitochondria, inhibits ASK1 kinaseactivity, and protects ECs from genotoxic stress. Mutation of the Raf-1activation domain (SS338/9AA) not only prevents Raf-1/ASK1 complexformation but also abolishes bFGF-mediated EC protection from genotoxicstress. Thus, selective type II inhibitors protein kinase, stabilizing aprotein kinase in the inactive state with a compound that are not ATPcompetitive inhibitors of the protein kinase in the active state, whichblock ASK1 at the mitochondria would prevent stress-mediated apoptosis.

Sensitizing a Cell to an Extrinsic Stress

In some embodiments, the methods described herein are used to sensitizea cell to an extrinsic stress comprising contacting a cell (before orafter exposing the cell to a stress) with a compound that (i) stabilizesthe protein kinase in the inactive state and (ii) is not an ATPcompetitive inhibitor of the protein kinase in the active state. Incertain embodiments, the stress is selected from hypoxia, chemotherapy,radiotherapy or glucose/nutrient starvation. In some embodiments, thecompound for the methods is a selective type II inhibitor; preferably aselective type II inhibitor of RAF kinase (e.g. B-RAF kinase); morepreferably the compound is also a selective type II inhibitor of a PDGFreceptor. In some embodiments, the compound modulates A-RAF. In otherembodiments, the compound for the methods inhibits theheterodimerization of BRAF with CRAF. In some embodiments, the compoundinhibits the phosphorylation of S338 of C-RAF. In another embodiment,the compound does not inhibit active kinases selected from the groupB-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, and PDGFRβ3. In someembodiments, the compound is an allosteric inhibitor of PDGFRα, PDGFRβ,Flt3 and/or c-Kit. In some embodiments, the compound has the structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

Combination Treatments

In general, the compositions described herein and, in embodiments wherecombinational therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition, and in someembodiments, because of different physical and chemical characteristics,are administered by different routes. In some embodiments, the initialadministration is made according to established protocols, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration is modified by the skilled clinician.

In some embodiments, therapeutically-effective dosages vary when thedevigs are used in treatment combinations. Combination treatment furtherincludes periodic treatments that start and stop at various times toassist with the clinical management of the patient. For combinationtherapies described herein, dosages of the co-administered compoundsvary depending on the type of co-drug employed, on the specific drugemployed, on the disease, disorder, or condition being treated and soforth.

It is understood that in some embodiments, the dosage regimen to treat,prevent, or ameliorate the condition(s) for which relief is sought, ismodified in accordance with a variety of factors. These factors includethe disorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, in otherembodiments, the dosage regimen actually employed varies widely andtherefore deviates from the dosage regimens set forth herein.

Combinations of compounds (i.e., allosteric kinase inhibitors describedherein) with other anti-cancer or chemotherapeutic agents are intendedto be covered. In some embodiments, examples of such agents are found inCancer Principles and Practice of Oncology by V. T. Devita and S.Hellman (editors), 6^(th) edition (Feb. 15, 2001), Lippincott Williams &Wilkins Publishers. Such anti-cancer agents include, but are not limitedto, the following: estrogen receptor modulators, androgen receptormodulators, retinoid receptor modulators, cytotoxic/cytostatic agents,antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoAreductase inhibitors, nitrogen mustards, nitroso ureas, angiogenesisinhibitors, inhibitors of cell proliferation and survival signalingpathway, apoptosis inducing agents, agents that interfere with cellcycle checkpoints, agents that interfere with receptor tyrosine kinases(RTKs), integrin blockers, NSAIDs, PPAR agonists, inhibitors of inherentmultidrug resistance (MDR), anti-emetic agents, agents useful in thetreatment of anemia, agents useful in the treatment of neutropenia,immunologic-enhancing drugs, biphosphonates, aromatase inhibitors,agents inducing terminal differentiation of neoplastic cells,γ-secretase inhibitors, cancer vaccines, and any combination thereof.

“Estrogen receptor modulators” refers to compounds that interfere orinhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpr-opanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

In some embodiments, estrogen receptor modulators are tamoxifen andraloxifene.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell mytosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, inhibitors of histonedeacetylase, inhibitors of kinases involved in mitotic progression,antimetabolites; biological response modifiers; hormonal/anti-hormonaltherapeutic agents, haematopoietic growth factors, monoclonal antibodytargeted therapeutic agents, topoisomerase inhibitors, proteasomeinhibitors and ubiquitin ligase inhibitors.

Examples of cytotoxic agents include, but are not limited to,tirapazimine, sertenef, cachectin, ifosfamide, tasonermin, lonidamine,carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine,fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin,estramustine, improsulfan tosilate, trofosfamide, nimustine,dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin,cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine,glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine-(chloro)platinum(II)]-tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

Examples of microtubulin inhibitors include paclitaxel, vindesinesulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol,rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin,RPR109881, BMS 184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)-benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide(SEQ ID NO: 2), TDX258, and BMS 188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)-ethyl]-(20S)camptothecin,BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide,sobuzoxane, 2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)colchic(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)-amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2-,1-c]quinolin-7-one, and dimesna.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxy-cytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]-glycylamino]-L-glycero-B-L-manno-heptopyranosyl]-adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester,swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, and3-aminopyridine-2-carboxaldehyde thiosemicarbazone. “Antiproliferativeagents” also includes monoclonal antibodies to growth factors, otherthan those listed under “angiogenesis inhibitors”, such as trastuzumab,and tumor suppressor genes, such as p53, which in some embodiments aredelivered via recombinant virus-mediated gene transfer.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude(±)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chloropheny-1)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(±)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethyl-phenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,(S)-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)-methyl)-2-piperazinone,5(S)-n-butyl-1-(2-methylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-yl-ethyl)carbamoyl]-piperidine,4-{5-[4-hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-[3-(2-oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxa-azacyclononadecine-9-carbonitrile,(±)-19,20-dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]-oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H, 17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4-h][1,8,11,14]oxatriazacyclo-eicosine-9-carbonitrile,and(±)-19,20-dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. In some embodiments, compoundswhich have inhibitory activity for HMG-CoA reductase are readilyidentified by using known assays. The terms “HMG-CoA reductaseinhibitor” and “inhibitor of HMG-CoA reductase” have the same meaningwhen used herein.

In some embodiments, examples of HMG-CoA reductase inhibitors that areused include but are not limited to lovastatin (MEVACOR®), simvastatin(ZOCOR®), pravastatin (PRAVACHOL®), fluvastatin (LESCOL®), atorvastatin(LIPITOR®) and cerivastatin (also known as rivastatin and BAYCHOL®). Insome embodiments, the structural formulas of these and additionalHMG-CoA reductase inhibitors that are used in the instant methods aredescribed at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”,Chemistry & Industry, pp. 85-89 (Feb. 5, 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefore the use of such salts, esters,open-acid and lactone forms is intended to be covered.

In some embodiments, in HMG-CoA reductase inhibitors where an open-acidform exists, salt and ester forms are formed from the open-acid, and allsuch forms are included within the meaning of the term “HMG-CoAreductase inhibitor” as used herein. In some embodiments, the HMG-CoAreductase inhibitor is selected from lovastatin and simvastatin. In oneembodiment, the HMG-CoA reductase inhibitor is simvastatin.

Herein, the term “pharmaceutically acceptable salts” with respect to theHMG-CoA reductase inhibitor shall mean non-toxic salts of the compoundsemployed which are generally prepared by reacting the free acid with asuitable organic or inorganic base, particularly those formed fromcations such as sodium, potassium, aluminum, calcium, lithium,magnesium, zinc and tetramethylammonium, as well as those salts formedfrom amines such as ammonia, ethylenediamine, N-methylglucamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1′-yl-methylbenzimidazole, diethylamine,piperazine, and tris(hydroxymethyl)aminomethane. In other embodiments,further examples of salt forms of HMG-CoA reductase inhibitors include,but are not limited to, acetate, benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, bromide, calcium hydroxyl,camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride,hydroxyl, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate,lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

In other embodiments, ester derivatives of the described HMG-CoAreductase inhibitor compounds act as prodrugs which, when absorbed intothe bloodstream of a warm-blooded animal, cleave in such a manner as torelease the drug form and permit the drug to afford improved therapeuticefficacy.

Examples of HIV protease inhibitors include amprenavir, abacavir,CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir,ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232, 632. Examples ofreverse transcriptase inhibitors include delaviridine, efavirenz,GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddl. It hasbeen reported that HIV protease inhibitors, such as indinavir orsaquinavir, have potent anti-angiogenic activities and promoteregression of Kaposi sarcoma.

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR20), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxygenase-2 inhibitors likecelecoxib, valecoxib, and rofecoxib, carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists, andantibodies to VEGF.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]-methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RP14610, NX31838, sulfatedmannopentose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonyl-imino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

“Inhibitors of cell proliferation and survival signaling pathway” referto pharmaceutical agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of inhibitors of EGFR (for example gefitinib anderlotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of CD20 (rituximab), inhibitors of cytokine receptors,inhibitors of MET, inhibitors of PDK (for example LY294002),serine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in (WO 03/086404, WO 03/086403, WO 03/086394, WO03/086279, WO 02/083675, WO 02/083139, WO 02/083140 and WO 02/083138),inhibitors of Raf kinase (for example BAY-43-9006), inhibitors of MEK(for example CI-1040 and PD-098059) and inhibitors of mTOR (for exampleWyeth CCI-779 and Ariad AP23573). Such agents include small moleculeinhibitor compounds and antibody antagonists.

“Apoptosis inducing agents” include, but not limited to, activators ofTNF receptor family members (including the TRAIL receptors).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer tocompounds that inhibit RTKs and therefore mechanisms involved inoncogenesis and tumor progression. Such agents include, but not limitedto, tyrosine kinase inhibitors such as inhibitors of c-Kit, Eph, PDGF,Flt3 and c-Met. Further agents include inhibitors of RTKs. Examplesof“tyrosine kinase inhibitors” include, but not limited to,N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]-quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein,ST1571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7-H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, SU11248,STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, andEMD121974.

Compounds (i.e., allosteric kinase inhibitors described herein) are alsouseful in combination with platelet fibrinogen receptor (GP Iib/IIIa)antagonists, such as tirofiban, to inhibit metastasis of cancerouscells. Tumor cells also activate platelets largely via thrombingeneration. This activation is associated with the release of VEGF. Therelease of VEGF enhances metastasis by increasing extravasation atpoints of adhesion to vascular endothelium (Amirkhosravi, 1999,Platelets 10: 285-292).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counter-act binding of a physiological ligand to the α_(v)β₅integrin, to compounds which antagonize, inhibit or counteract bindingof a physiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆;α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of thecompounds (i.e., allosteric kinase inhibitors described herein) withPPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta)agonists are useful in the treatment of certain malignancies. PPAR-γ andPPAR-δ are the nuclear peroxisome proliferator-activated receptors γ andδ. More recently, PPAR-γ agonists have been shown to inhibit theangiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. Examples of PPAR-γ agonists and PPAR-γ/αagonists include, but are not limited to, thiazolidinediones (such asDRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone),fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242,JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NPOl 10, DRF4158,NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

In some embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are used in combination with gene therapy for thetreatment of cancer. In other embodiments, gene therapy is used todeliver any tumor suppressing gene. Examples of such genes include, butare not limited to, p53, which in some embodiments is delivered viarecombinant virus-mediated gene transfer, Duc-4, NF-I, NF-2, RB, WT1,BRCA1, BRCA2, a uPA/uPAR antagonist.

In other embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are also administered in combination with an inhibitorof inherent multidrug resistance (MDR), in particular MDR associatedwith high levels of expression of transporter proteins. Such MDRinhibitors include inhibitors of p-glycoprotein (P-gp), such asLY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).

In some embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are employed in conjunction with anti-emetic agents totreat nausea or emesis, including acute, delayed, late-phase, andanticipatory emesis, which result from use of the compounds (i.e.,allosteric kinase inhibitors described herein), alone or with radiationtherapy. In further embodiments, for the prevention or treatment ofemesis, compounds (i.e., allosteric kinase inhibitors described herein)are used in conjunction with other anti-emetic agents, especiallyneurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such asondansetron, granisetron, tropisetron, and zatisetron, GABAB receptoragonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that results upon administration ofthe instant compounds.

In other embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are also administered with an agent useful in thetreatment of anemia. Such an anemia treatment agent is, for example, acontinuous eythropoiesis receptor activator (such as epoetin-α).

In further embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are also administered with an agent usefulin the treatment of neutropenia. Such a neutropenia treatment agent is,for example, a hematopoietic growth factor which regulates theproduction and function of neutrophils such as a human granulocytecolony stimulating factor, (G-CSF). Examples of a G-CSF includefilgrastim.

In some embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are also administered with an immunologic-enhancingdrug, such as levamisole, bacillus Calmette-Guerin, octreotide,isoprinosine and Zadaxin.

In other embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are also useful for treating or preventing cancer,including bone cancer, in combination with bisphosphonates (understoodto include bisphosphonates, diphosphonates, bisphosphonic acids anddiphosphonic acids). Examples of bisphosphonates include but are notlimited to: etidronate (Didronel), pamidronate (Aredia), alendronate(Fosamax), risedronate(Actonel), zoledronate (Zometa), ibandronate(Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate,neridronate, piridronate and tiludronate including any and allpharmaceutically acceptable salts, derivatives, hydrates and mixturesthereof.

In further embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are also useful for treating or preventingbreast cancer in combination with aromatase inhibitors. Examples ofaromatase inhibitors include but are not limited to: anastrozole,letrozole and exemestane.

In some embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are also useful for treating or preventing cancer incombination with siRNA or RNAi therapeutics.

In some other embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are also useful for treating or preventingcancer in combination with compounds which induce terminaldifferentiation of the neoplastic cells. Suitable differentiation agentsinclude the following compounds: (a) Polar compounds; (b) Derivatives ofvitamin D and retinoic acid; (c) Steroid hormones; (d) Growth factors;(e) Proteases; (f) Tumor promoters; and (g) inhibitors of DNA or RNAsynthesis.

“DNA methyltransferase inhibitor” refers to compounds which inhibit themethylation of the DNA base cytosine at the C-5 position of that base bythe DNA methyltransferase enzyme. Examples of such DNA methyltransferaseinhibitor include DNA methyltransferase inhibitors include 5-azacytosineand Zebularine®.

Examples of an antineoplastic agent include, in general,microtubule-stabilizing agents (such as paclitaxel (also known asTaxol®), docetaxel (also known as Taxotere®, epothilone A, epothilone B,desoxyepothilone A, desoxyepothilone B or their derivatives);microtubule-disruptor agents; alkylating agents, anti-metabolites;epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes; biologicalresponse modifiers and growth inhibitors; hormonal/anti-hormonaltherapeutic agents and haematopoietic growth factors.

Example classes of antineoplastic agents include, for example, theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the taxanes, the epothilones,discodermolide, the pteridine family of drugs, diynenes and thepodophyllotoxins. Particularly useful members of those classes include,for example, doxorubicin, carminomycin, daunorubicin, aminopterin,methotrexate, methopterin, dichloro-methotrexate, mitomycin C,porfiromycin, Herceptin®, Rituxan®, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podo-phyllotoxinderivatives such as colchicines, etoposide, etoposide phosphate orteniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine,leurosine, paclitaxel and the like. Other useful antineoplastic agentsinclude estramustine, cisplatin, carboplatin, cyclophosphamide,bleomycin, tamoxifen, ifosamide, melphalan, hexamethyl melamine,thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine,L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide,flutamide, leuprolide, pyridobenzoindole derivatives, interferons andinterleukins. In some embodiments, the antineoplastic agents are thetaxanes and the antineoplastic agent is paclitaxel.

Radiation Therapy

In some embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are administered in combination with radiotherapy.Radiotherapy, also called radiation therapy, is the treatment of cancerand other diseases with ionizing radiation. Ionizing radiation depositsenergy that injures or destroys cells in an area being treated (a“target tissue”) by damaging their genetic material, making itimpossible for these cells to continue to grow. Although radiationdamages both cancer cells and normal cells, the latter are better ableto repair themselves and function properly. In some embodiments,radiotherapy is used to treat localized solid tumors, such as cancers ofthe skin, tongue, larynx, brain, breast, prostate, colon, uterus and/orcervix. In some embodiments, it is also used to treat leukemia andlymphoma (cancers of the blood-forming cells and lymphatic system,respectively).

One type of radiation therapy commonly used involves photons, “packets”of energy. X-rays, gamma rays are both photon radiation to be used totreat cancer. Depending on the amount of energy they possess, the raysare used to destroy cancer cells on the surface of or deeper in thebody. In other embodiments, the higher the energy of the ray beam, thedeeper the rays go into the target tissue.

Another technique for delivering radiation to cancer cells is to placeradioactive implants directly in a tumor or body cavity. This is calledinternal radiotherapy (brachytherapy, interstitial irradiation, andintracavitary irradiation are types of internal radiotherapy.) Usinginternal radiotherapy, the radiation dose is concentrated in a smallarea, and the patient stays in the hospital for a few days. Internalradiotherapy is frequently used for cancers of the tongue, uterus,prostate, colon, and cervix.

The term “radiotherapy” or “ionizing radiation” includes all forms ofradiation, including but not limited to α, β, and γ radiation and ultraviolet light, which are capable of directly or indirectly damaging thegenetic material of a cell or virus. The term “irradiation” refers tothe exposure of a sample of interest to ionizing radiation. Radiotherapywith or without concurrent or sequential chemotherapy is an effectivemodality for head and neck, breast, skin, anogenital cancers, andcertain nonmalignant diseases such as keloid, desmoid tumor, hemangioma,arteriovenous malformation, and histocytosis X. However, the therapeuticbenefit is limited by radiation- and chemotherapy-induced mucosalepithelium injuries and cutaneous radiation syndrome (CRS), whichinclude acute reactions of tissue swelling, mucositis, dermatitis,desquamation, and ulceration, and long-term effects of tissue/skinfibrosis, necrosis, and the development of life-threatening sequelae ofsarcoma, squamous and basal cell carcinoma.

MEK1/2- and/or ERK1/2-Mediated Cellular Proliferation or Migration

In other embodiments, the methods described here for inhibiting MEK1/2-and/or ERK1/2-mediated cellular proliferation or migration comprisecontacting a cell with a compound that (i) stabilizes the protein kinasein the inactive state and (ii) is not an ATP competitive inhibitor ofthe protein kinase in the active state. In certain embodiments, thecompound blocks VEGF- and/or FGF-stimulated endothelial responses intumor angiogenesis. In some embodiments, the compound for the methods isa selective type II inhibitor; preferably a selective type II inhibitorof RAF kinase (e.g. a B-RAF kinase); more preferably the compound isalso a selective type II inhibitor of a PDGF receptor. In otherembodiments, the compound for the methods inhibits theheterodimerization of BRAF with CRAF. In some embodiments, the compoundinhibits the phosphorylation of S338 of C-RAF. In another embodiment,the compound does not inhibit active kinases selected from the groupB-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, and PDGFRβ3. In someembodiments, the compound is an allosteric inhibitor of PDGFRα, PDGFRβ,Flt3 and/or c-Kit. In some embodiments, the compound has the structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

The MAPK/ERK pathway is a signal transduction pathway that couplesintracellular responses to the binding of growth factors to cell surfacereceptors. Receptor-linked tyrosine kinases such as the epidermal growthfactor receptor (EGFR) are activated by extracellular ligands. Bindingof epidermal growth factor (EGF) to the EGFR activates the tyrosinekinase activity of the cytoplasmic domain of the receptor. The EGFRbecomes phosphorylated on tyrosine residues. The basic pathway shown inthe figure describes MAPK signaling in cells depending on proteinscaffold formation and assembly of complex between B- and C-RAF leadingto MEK/ERK activation.

Disruption of B-/C-RAF complex thus inhibits MARK signaling that leadsto suppression of angiogenesis. Thus, by employing a compound that (i)stabilizes the protein kinase in the inactive state and (ii) is not anATP competitive inhibitor of the protein kinase in the active state,inhibition of MEK1/2- and/or ERK1/2-mediated cellular proliferation ormigration can be realized.

Treatment of Restenosis

In some embodiments, the present invention provides methods for treatingrestenosis in a human subject comprising administering to a patient inneed a compound that (i) stabilizes a protein kinase in the inactivestate and (ii) is not an ATP competitive inhibitor of the protein kinasein the active state. In certain embodiments, the restenosis is intimalhyperplasia-driven restenosis after vascular injury. In someembodiments, the compound for the methods is a selective type IIinhibitor of RAF kinase (e.g. B-RAF kinase); more preferably thecompound is also a selective type II inhibitor of a PDGF receptor. Insome embodiments, the compound modulates A-RAF. In other embodiments,the compound for the methods inhibits the heterodimerization of BRAFwith CRAF. In some embodiments, the compound inhibits thephosphorylation of S338 of C-RAF. In another embodiment, the compounddoes not inhibit active kinases selected from the group B-RAF, C-RAF,VEGFR1, VEGFR2, Flt3, Kit, and PDGFRβ. In some embodiments, the compoundis an allosteric inhibitor of PDGFRα, PDGFRβ, Flt3 and/or c-Kit. In someembodiments, the compound has the structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

Restenosis literally means the reoccurrence of stenosis, a narrowing ofa blood vessel, leading to restricted blood flow. Restenosis usuallyrefers to an artery or other large blood vessel that has becomenarrowed, received treatment to clear the blockage and subsequentlybecome renarrowed. This is usually restenosis of an artery, or otherblood vessel, or possibly a vessel within an organ. Restenosis commonlyresults from balloon angioplasty and/or stent placement resulting ineventual occlusion of arteries by a process described as neointimalhyperplasia (NIH). After arterial injury, an over-proliferation ofvascular smooth muscle cells occurs which has previously been shown tobe dependent on both PDGFR/3P (Englesbe, et al. (2004) J Vasc Surg 39,440-6) and MAPK pathway activation (Li, et al. (2005) Circulation 111,1672-8; Pintucci, et al. (2006) Faseb J 20, 398-400). Therefore, thecombination of PDGFRβ/RAF inhibition would be an ideal treatment forNIH; as such, provided herein compounds that (i) stabilizes a proteinkinase in the inactive state and (ii) is not an ATP competitiveinhibitor of the protein kinase in the active state which may beselected PDGFRβ/RAF inhibitors.

Treatment of Fibrosis

Tissue injury initiates a complex series of host wound-healingresponses; if successful, these responses restore normal tissuestructure and function. If not, these responses can lead to tissuefibrosis and loss of function.

In other embodiments, the methods described here for treating fibroticdiseases in a human subject comprise administering to a patient in needa compound that (i) stabilizes a protein kinase in the inactive stateand (ii) is not an ATP competitive inhibitor of the protein kinase inthe active state. In certain embodiments, the fibrosis is pulmonaryfibrosis. In certain embodiments, the fibrosis is liver fibrosis. Insome embodiments, the compound for the methods is a selective type IIinhibitor; preferably a selective type II inhibitor of RAF kinase (e.g.B-RAF kinase); more preferably the compound is also a selective type IIinhibitor of a PDGF receptor. In some embodiments, the compound for themethods modulates A-RAF. In other embodiments, the compound for themethods inhibits the heterodimerization of BRAF with CRAF. In someembodiments, the compound inhibits the phosphorylation of S338 of C-RAF.In another embodiment, the compound does not inhibit active kinasesselected from the group B-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, andPDGFRβ. In some embodiments, the compound is an allosteric inhibitor ofPDGFRα, PDGFRβ, Flt3 and/or c-Kit. In some embodiments, the compound hasthe structure

wherein R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted amine, NH₂, optional substitutedalkyoxy, optional substituted thioalkyl, CF₃S, optional substitutedalkylsulfinyl or optional substituted alkylsulfonyl; Z is NH, S or O; Z′is N or C; and Ar is phenyl or bicyclic phenyl optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, andC₃₋₁₀cycloalkyl. In certain embodiments, the compound has the structure

wherein R^(m) is C₁₋₆ alkyl, or halogen substituted C₁₋₆ alkyl; and R₃is independently a hydrogen, C₁₋₆ alkyl, halogen substituted C₁₋₆ alkyl,halogen substituted C₁₋₆ alkoxy, or C₃₋₁₀cycloalkyl; or, optionally,R^(m) and R₃ are joined to form a five to seven membered carbocycle; andn is 0-4. In certain embodiments, the compound has the structure

wherein R₃ is hydrogen or halogen; and Z is NH, S or O. In anotherembodiment, the compound is selected from

The terms “fibrosis” or “fibrosing disorder,” as used herein, refers toconditions that are associated with the abnormal accumulation of cellsand/or fibronectin and/or collagen and/or increased fibroblastrecruitment and include but are not limited to fibrosis of individualorgans or tissues such as the heart, kidney, liver, joints, lung,pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletaland digestive tract.

Exemplary diseases, disorders, or conditions that involve fibrosisinclude, but are not limited to: Lung diseases associated with fibrosis,e.g., idiopathic pulmonary fibrosis, pulmonary fibrosis secondary tosystemic inflammatory disease such as rheumatoid arthritis, scleroderma,lupus, cryptogenic fibrosing alveolitis, radiation induced fibrosis,chronic obstructive pulmonary disease (COPD), scleroderma, chronicasthma, silicosis, asbestos induced pulmonary or pleural fibrosis, acutelung injury and acute respiratory distress (including bacterialpneumonia induced, trauma induced, viral pneumonia induced, ventilatorinduced, non-pulmonary sepsis induced, and aspiration induced); Chronicnephropathies associated with injury/fibrosis (kidney fibrosis), e.g.,glomerulonephritis secondary to systemic inflammatory diseases such aslupus and scleroderma, diabetes, glomerular nephritis, focal segmentalglomerular sclerosis, IgA nephropathy, hypertension, allograft andAlport; Gut fibrosis, e.g., scleroderma, and radiation induced gutfibrosis; Liver fibrosis, e.g., cirrhosis, alcohol induced liverfibrosis, nonalcoholic steatohepatitis (NASH), biliary duct injury,primary biliary cirrhosis, infection or viral induced liver fibrosis(e.g., chronic HCV infection), and autoimmune hepatitis; Head and neckfibrosis, e.g., radiation induced; Corneal scarring, e.g., LASIK(laser-assisted in situ keratomileusis), corneal transplant, andtrabeculectomy; Hypertrophic scarring and keloids, e.g., burn induced orsurgical; and Other fibrotic diseases, e.g., sarcoidosis, scleroderma,spinal cord injury/fibrosis, myelofibrosis, vascular restenosis,atherosclerosis, arteriosclerosis, Wegener's granulomatosis, mixedconnective tissue disease, and Peyronie's disease.

In one aspect, compounds (i.e., allosteric kinase inhibitors describedherein) are administered to a mammal with fibrosis of an organ or tissueor with a predisposition of developing fibrosis of an organ or tissuewith one or more other agents that are used to treat fibrosis. In oneaspect, the one or more agents include corticosteroids. In one aspect,the one or more agents include immunosuppresants. In one aspect, the oneor more agents include B-cell antagonists. In one aspect, the one ormore agents include uteroglobin.

Certain Pharmaceutical and Medical Terminology

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The term “antagonist,” as used herein, refers to a molecule such as acompound, which diminishes, inhibits, or prevents the action of anothermolecule or the activity of a receptor site. Antagonists include, butare not limited to, competitive antagonists, non-competitiveantagonists, uncompetitive antagonists, partial agonists and inverseagonists.

Competitive antagonists reversibly bind to receptors at the same bindingsite (active site) as the endogenous ligand or agonist, but withoutactivating the receptor.

Allosteric inhibitors (also known as non-competitive antagonists) bindto a distinctly separate binding site from the agonist, exerting theiraction to that receptor via the other binding site. Non-competitiveantagonists do not compete with agonists for binding. The boundantagonists may result in a decreased affinity of an agonist for thatreceptor, or alternatively may prevent conformational changes in thereceptor required for receptor activation after the agonist binds.

Uncompetitive antagonists differ from non-competitive antagonists inthat they require receptor activation by an agonist before they can bindto a separate allosteric binding site.

The term “cancer,” as used herein refers to an abnormal growth of cellswhich tend to proliferate in an uncontrolled way and, in some cases, tometastasize (spread). The types of cancer include, but is not limitedto, solid tumors (such as those of the bladder, bowel, brain, breast,endometrium, heart, kidney, lung, lymhatic tissue (lymphoma), ovary,pancreas or other endocrine organ (thyroid), prostate, skin (melanoma)or hematological tumors (such as the leukemias).

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case may be determinedusing techniques, such as a dose escalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “kit” and “article of manufacture” are used as synonyms.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Metabolites of the compounds disclosed herein areoptionally identified either by administration of compounds to a hostand analysis of tissue samples from the host, or by incubation ofcompounds with hepatic cells in vitro and analysis of the resultingcompounds.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound (i.e., allosteric kinase inhibitorsdescribed herein) and a co-agent, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. acompound (i.e., allosteric kinase inhibitors described herein) and aco-agent, are administered to a patient as separate entities eithersimultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

The term “pharmaceutical composition” refers to a mixture of a compound(i.e., allosteric kinase inhibitors described herein) with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one embodiment, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Routes of Administration

Suitable routes of administration include, but are not limited to, oral,intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary,transmucosal, transdermal, vaginal, otic, nasal, and topicaladministration. In addition, by way of example only, parenteral deliveryincludes intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, a compound as described herein is administeredin a local rather than systemic manner, for example, via injection ofthe compound directly into an organ, often in a depot preparation orsustained release formulation. In specific embodiments, long actingformulations are administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection.Furthermore, in other embodiments, the drug is delivered in a targeteddrug delivery system, for example, in a liposome coated withorgan-specific antibody. In such embodiments, the liposomes are targetedto and taken up selectively by the organ. In yet other embodiments, thecompound as described herein is provided in the form of a rapid releaseformulation, in the form of an extended release formulation, or in theform of an intermediate release formulation. In yet other embodiments,the compound described herein is administered topically.

Pharmaceutical Composition/Formulation

In some embodiments, the compounds described herein are formulated intopharmaceutical compositions. In specific embodiments, pharmaceuticalcompositions are formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any pharmaceuticallyacceptable techniques, carriers, and excipients are used as suitable toformulate the pharmaceutical compositions described herein: Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999).

Provided herein are pharmaceutical compositions comprising a compound(i.e., allosteric kinase inhibitors described herein) and apharmaceutically acceptable diluent(s), excipient(s), or carrier(s). Incertain embodiments, the compounds described are administered aspharmaceutical compositions in which a compound (i.e., allosteric kinaseinhibitors described herein) is mixed with other active ingredients, asin combination therapy. Encompassed herein are all combinations ofactives set forth in the combination therapies section below andthroughout this disclosure. In specific embodiments, the pharmaceuticalcompositions include one or more compounds (i.e., allosteric kinaseinhibitors described herein).

A pharmaceutical composition, as used herein, refers to a mixture of acompound (i.e., allosteric kinase inhibitors described herein) withother chemical components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. In certain embodiments, the pharmaceutical compositionfacilitates administration of the compound to an organism. In someembodiments, practicing the methods of treatment or use provided herein,therapeutically effective amounts of compounds (i.e., allosteric kinaseinhibitors described herein) are administered in a pharmaceuticalcomposition to a mammal having a disease or condition to be treated. Inspecific embodiments, the mammal is a human. In certain embodiments,therapeutically effective amounts vary depending on the severity of thedisease, the age and relative health of the subject, the potency of thecompound used and other factors. The compounds described herein are usedsingly or in combination with one or more therapeutic agents ascomponents of mixtures.

In one embodiment, a compound (i.e., allosteric kinase inhibitorsdescribed herein) is formulated in an aqueous solution. In specificembodiments, the aqueous solution is selected from, by way of exampleonly, a physiologically compatible buffer, such as Hank's solution,Ringer's solution, or physiological saline buffer. In other embodiments,a compound (i.e., allosteric kinase inhibitors described herein) isformulated for transmucosal administration. In specific embodiments,transmucosal formulations include penetrants that are appropriate to thebarrier to be permeated. In still other embodiments wherein thecompounds described herein are formulated for other parenteralinjections, appropriate formulations include aqueous or nonaqueoussolutions. In specific embodiments, such solutions includephysiologically compatible buffers and/or excipients.

In another embodiment, compounds described herein are formulated fororal administration. Compounds described herein, including a compound(i.e., allosteric kinase inhibitors described herein), are formulated bycombining the active compounds with, e.g., pharmaceutically acceptablecarriers or excipients. In various embodiments, the compounds describedherein are formulated in oral dosage forms that include, by way ofexample only, tablets, powders, pills, dragees, capsules, liquids, gels,syrups, elixirs, slurries, suspensions and the like.

In certain embodiments, pharmaceutical preparations for oral use areobtained by mixing one or more solid excipient with one or more of thecompounds described herein, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as:for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Inspecific embodiments, disintegrating agents are optionally added.Disintegrating agents include, by way of example only, cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

In one embodiment, dosage forms, such as dragee cores and tablets, areprovided with one or more suitable coating. In specific embodiments,concentrated sugar solutions are used for coating the dosage form. Thesugar solutions, optionally contain additional components, such as byway of example only, gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs and/orpigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionallyutilized to characterize different combinations of active compounddoses.

In certain embodiments, therapeutically effective amounts of at leastone of the compounds described herein are formulated into other oraldosage forms. Oral dosage forms include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. In specific embodiments,push-fit capsules contain the active ingredients in admixture with oneor more filler. Fillers include, by way of example only, lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In other embodiments, softcapsules, contain one or more active compound that is dissolved orsuspended in a suitable liquid. Suitable liquids include, by way ofexample only, one or more fatty oil, liquid paraffin, or liquidpolyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, therapeutically effective amounts of at least oneof the compounds described herein are formulated for buccal orsublingual administration. Formulations suitable for buccal orsublingual administration include, by way of example only, tablets,lozenges, or gels. In still other embodiments, the compounds describedherein are formulated for parental injection, including formulationssuitable for bolus injection or continuous infusion. In specificembodiments, formulations for injection are presented in unit dosageform (e.g., in ampoules) or in multi-dose containers. Preservatives are,optionally, added to the injection formulations. In still otherembodiments, the pharmaceutical compositions of a compound (i.e.,allosteric kinase inhibitors described herein) are formulated in a formsuitable for parenteral injection as a sterile suspensions, solutions oremulsions in oily or aqueous vehicles. Parenteral injection formulationsoptionally contain formulatory agents such as suspending, stabilizingand/or dispersing agents. In specific embodiments, pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. In additional embodiments,suspensions of the active compounds are prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles for usein the pharmaceutical compositions described herein include, by way ofexample only, fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. In certainspecific embodiments, aqueous injection suspensions contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension contains suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Alternatively, in other embodiments, the activeingredient is in powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

In one aspect, compounds (i.e., allosteric kinase inhibitors describedherein) are prepared as solutions for parenteral injection as describedherein or known in the art and administered with an automatic injector.Automatic injectors, such as those disclosed in U.S. Pat. Nos.4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and WO/2005/087297(each of which are incorporated herein by reference for such disclosure)are known. In general, all automatic injectors contain a volume ofsolution that includes a compound (i.e., allosteric kinase inhibitorsdescribed herein) to be injected. In general, automatic injectorsinclude a reservoir for holding the solution, which is in fluidcommunication with a needle for delivering the drug, as well as amechanism for automatically deploying the needle, inserting the needleinto the patient and delivering the dose into the patient. Exemplaryinjectors provide about 0.3 mL of solution at about a concentration of0.5 mg to 10 mg of a compound (i.e., allosteric kinase inhibitorsdescribed herein) per 1 mL of solution. Each injector is capable ofdelivering only one dose of the compound.

In still other embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are administered topically. The compoundsdescribed herein are formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compositions optionally contain solubilizers,stabilizers, tonicity enhancing agents, buffers and preservatives.

In yet other embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are formulated for transdermaladministration. In specific embodiments, transdermal formulations employtransdermal delivery devices and transdermal delivery patches and can belipophilic emulsions or buffered, aqueous solutions, dissolved and/ordispersed in a polymer or an adhesive. In various embodiments, suchpatches are constructed for continuous, pulsatile, or on demand deliveryof pharmaceutical agents. In additional embodiments, the transdermaldelivery of a compound (i.e., allosteric kinase inhibitors describedherein) is accomplished by means of iontophoretic patches and the like.In certain embodiments, transdermal patches provide controlled deliveryof a compound (i.e., allosteric kinase inhibitors described herein). Inspecific embodiments, the rate of absorption is slowed by usingrate-controlling membranes or by trapping the compound within a polymermatrix or gel. In alternative embodiments, absorption enhancers are usedto increase absorption. Absorption enhancers or carriers includeabsorbable pharmaceutically acceptable solvents that assist passagethrough the skin. For example, in one embodiment, transdermal devicesare in the form of a bandage comprising a backing member, a reservoircontaining the compound optionally with carriers, optionally a ratecontrolling barrier to deliver the compound to the skin of the host at acontrolled and predetermined rate over a prolonged period of time, andmeans to secure the device to the skin.

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiment, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound(i.e., allosteric kinase inhibitors described herein); (2) a penetrationenhancer; and (3) an aqueous adjuvant. In addition, transdermalformulations can include additional components such as, but not limitedto, gelling agents, creams and ointment bases, and the like. In someembodiments, the transdermal formulation further include a woven ornon-woven backing material to enhance absorption and prevent the removalof the transdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein maintain a saturated orsupersaturated state to promote diffusion into the skin.

In other embodiments, the compounds (i.e., allosteric kinase inhibitorsdescribed herein) are formulated for administration by inhalation.Various forms suitable for administration by inhalation include, but arenot limited to, aerosols, mists or powders. Pharmaceutical compositionsof a compound (i.e., allosteric kinase inhibitors described herein) areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Inspecific embodiments, the dosage unit of a pressurized aerosol isdetermined by providing a valve to deliver a metered amount. In certainembodiments, capsules and cartridges of, such as, by way of exampleonly, gelatin for use in an inhaler or insufflator are formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated by reference. Formulations, whichinclude a compound (i.e., allosteric kinase inhibitors describedherein), which are prepared according to these and other techniqueswell-known in the art are prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, fluorocarbons, and/orother solubilizing or dispersing agents known in the art. See, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are found in sources such asREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005, astandard reference in the field. The choice of suitable carriers ishighly dependent upon the exact nature of the nasal dosage form desired,e.g., solutions, suspensions, ointments, or gels. Nasal dosage formsgenerally contain large amounts of water in addition to the activeingredient. Minor amounts of other ingredients such as pH adjusters,emulsifiers or dispersing agents, preservatives, surfactants, gellingagents, or buffering and other stabilizing and solubilizing agents mayalso be present. Preferably, the nasal dosage form should be isotonicwith nasal secretions.

For administration by inhalation, the compounds described herein, may bein a form as an aerosol, a mist or a powder. Pharmaceutical compositionsdescribed herein are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

In still other embodiments, the compounds (i.e., allosteric kinaseinhibitors described herein) are formulated in rectal compositions suchas enemas, rectal gels, rectal foams, rectal aerosols, suppositories,jelly suppositories, or retention enemas, containing conventionalsuppository bases such as cocoa butter or other glycerides, as well assynthetic polymers such as polyvinylpyrrolidone, PEG, and the like. Insuppository forms of the compositions, a low-melting wax such as, butnot limited to, a mixture of fatty acid glycerides, optionally incombination with cocoa butter is first melted.

In certain embodiments, pharmaceutical compositions are formulated inany conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any pharmaceutically acceptable techniques,carriers, and excipients is optionally used as suitable and asunderstood in the art. Pharmaceutical compositions comprising a compound(i.e., allosteric kinase inhibitors described herein) may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

Pharmaceutical compositions include at least one pharmaceuticallyacceptable carrier, diluent or excipient and at least one compound(i.e., allosteric kinase inhibitors described herein) described hereinas an active ingredient. The active ingredient is in free-acid orfree-base form, or in a pharmaceutically acceptable salt form. Inaddition, the methods and pharmaceutical compositions described hereininclude the use of N-oxides, crystalline forms (also known aspolymorphs), as well as active metabolites of these compounds having thesame type of activity. All tautomers of the compounds described hereinare included within the scope of the compounds presented herein.Additionally, the compounds described herein encompass unsolvated aswell as solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like. The solvated forms of the compoundspresented herein are also considered to be disclosed herein. Inaddition, the pharmaceutical compositions optionally include othermedicinal or pharmaceutical agents, carriers, adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure, buffers, and/orother therapeutically valuable substances.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically acceptable excipients or carriers to form asolid, semi-solid or liquid. Solid compositions include, but are notlimited to, powders, tablets, dispersible granules, capsules, cachets,and suppositories. Liquid compositions include solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, but are notlimited to, gels, suspensions and creams. The form of the pharmaceuticalcompositions described herein include liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions also optionally contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and so forth.

In some embodiments, pharmaceutical composition comprising at leastcompound (i.e., allosteric kinase inhibitors described herein)illustratively takes the form of a liquid where the agents are presentin solution, in suspension or both. Typically when the composition isadministered as a solution or suspension a first portion of the agent ispresent in solution and a second portion of the agent is present inparticulate form, in suspension in a liquid matrix. In some embodiments,a liquid composition includes a gel formulation. In other embodiments,the liquid composition is aqueous.

In certain embodiments, pharmaceutical aqueous suspensions include oneor more polymers as suspending agents. Polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Certain pharmaceutical compositionsdescribed herein include a mucoadhesive polymer, selected from, forexample, carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

Pharmaceutical compositions also, optionally include solubilizing agentsto aid in the solubility of a compound (i.e., allosteric kinaseinhibitors described herein). The term “solubilizing agent” generallyincludes agents that result in formation of a micellar solution or atrue solution of the agent. Certain acceptable nonionic surfactants, forexample polysorbate 80, are useful as solubilizing agents, as canophthalmically acceptable glycols, polyglycols, e.g., polyethyleneglycol 400, and glycol ethers.

Furthermore, pharmaceutical compositions optionally include one or morepH adjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

Additionally, pharmaceutical compositions optionally include one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Other pharmaceutical compositions optionally include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

Still other pharmaceutical compositions include one or more surfactantsto enhance physical stability or for other purposes. Suitable nonionicsurfactants include polyoxyethylene fatty acid glycerides and vegetableoils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

Still other pharmaceutical compositions may include one or moreantioxidants to enhance chemical stability where required. Suitableantioxidants include, by way of example only, ascorbic acid and sodiummetabisulfite.

In certain embodiments, pharmaceutical aqueous suspension compositionsare packaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers are used, in which case it istypical to include a preservative in the composition.

In alternative embodiments, other delivery systems for hydrophobicpharmaceutical compounds are employed. Liposomes and emulsions areexamples of delivery vehicles or carriers herein. In certainembodiments, organic solvents such as N-methylpyrrolidone are alsoemployed. In additional embodiments, the compounds described herein aredelivered using a sustained-release system, such as semipermeablematrices of solid hydrophobic polymers containing the therapeutic agent.Various sustained-release materials are useful herein. In someembodiments, sustained-release capsules release the compounds for a fewhours up to over 24 hours. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed.

In certain embodiments, the formulations described herein include one ormore antioxidants, metal chelating agents, thiol containing compoundsand/or other general stabilizing agents. Examples of such stabilizingagents, include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Assays and Clinical Management

In certain embodiments, provided here are methods for identifying anallosteric inhibitor of RAF kinase comprising contacting a BRAF kinasewith a test compound and monitoring the phosphorylation of S338 of CRAF,wherein a decrease in the phosphorylation of S338 of CRAF relative tonon-contacted BRAF kinase indicates that the test compound is anallosteric inhibitor of BRAF kinase. In certain embodiments, theallosteric inhibitor of RAF kinase is a allosteric inhibitor of BRAFkinase.

In certain embodiments, provided here are methods for identifying atherapeutic agent for treating cancer comprising contacting a RAF kinase(e.g. a BRAF) with a test compound and monitoring the phosphorylation ofS338 of CRAF, wherein a decrease in the phosphorylation of S338 of CRAFrelative to non-contacted BRAF kinase indicates that the test compoundis capable of treating cancer.

In some embodiments provided here are methods for monitoring patientpharmacologic response in a chemotherapic treatment comprisingmonitoring the phosphorylation of S338 of CRAF, wherein a decrease inthe phosphorylation of S338 of CRAF relative to non-contacted BRAFkinase indicates that the chemoehtrapic treatment has positivepharmacologic response.

In some embodiments provide uses of pS338-CRAF as a predictive biomarkerfor clinical management of a cancer treatment. In certain embodiments, adecrease in the phosphorylation of S338 of CRAF relative tonon-contacted RAF kinase indicates a likelihood of benefit from saidcancer treatment. Biomarkers can be pre-treatment measurements used tocharacterize the patient's disease state in order to determine whetherthe patient is a good candidate for certain treatments. These biomarkersare called predictive biomarkers. As shown in the disclosure here, thephosphorylation of S338 of CRAF relative to non-contacted RAF kinasecorrelate to cancer proliferation. RAF ATP-mimetics induce RAFpS338-CRAF and thus induce RAF dimer formation in cancer cells. Use ofpS338-CRAF as a predictive biomarker thus can provide clinical cancertreatment management.

Type II inhibitors represent a new paradigm for drug selection as seenwith the co-crystal structures of Abl with imatinib (1) and B-RAF withsorafenib (2). The approach of stabilizing the inactive kinaseconformation has led to the development of novel inhibitors whichstabilize the DFG-out conformation (Liu, et al. (2006) Nature chemicalbiology 2:358-364, Okram, et al. (2006) Chemistry & biology 13:779-786).As discussed before, mutation of the Raf-1 activation domain (SS338/9AA)not only prevents Raf-1/ASK1 complex formation but also abolishesbFGF-mediated EC protection from genotoxic stress. Thus, by contacting aBRAF kinase with a test compound and monitoring the phosphorylation ofS338 of CRAF, wherein a decrease in the phosphorylation of S338 of CRAFrelative to non-contacted BRAF kinase would indicate that the testcompound is an allosteric inhibitor of BRAF kinase.

EXAMPLES

Animal Studies.

All animal procedures were conducted in accordance with all appropriateregulatory standards under protocols #S05018 and #S06008 approved by theUniversity of California, San Diego Institutional Animal Care and UseCommittee.

Computational Docking Studies and Chemical Synthesis.

The molecular modeling and homology model of PDGFRβ are described asfollows. All compounds were synthesized from commercially availablestarting materials and schemes, synthetic procedures, and purificationdetails are available as follows.

The x-ray crystal structure of B-RAF with BAY43-9006 (sorafenib) wasselected for docking studies since it contained the DFG motif of theactivation loop in a desirable inactive state (i.e. DFG-out). Moreoverthis inactive state is stabilized by a small molecule and as a resultprovides an appropriate ligand-induced conformation for docking with 6.The Docking module of InsightII (Accelrys, San Diego, Calif.) was usedto perform ligand docking studies with the previously reportedco-crystal structure of B-RAF with BAY 43-9006 (sorafenib). Thisinactive conformation was chosen since stabilizing the DFG out structureis important for the type II kinase inhibition mode. As a measure ofdocking accuracy the ligand from the crystal structure, BAY43-9006, wasremoved and docking from a 2D structure of this molecule was performedand qualitatively interpreted. The homology model of the PDGFRβ kinasemodule was created based on the crystal structure of VEGFR2 (pdb ID1Y6B). VEGFR2 and PDGFRβ share 46% sequence identity within the kinasedomain. Homology modeling was performed using the Homology module of theInsightII program (Accelrys, San Diego, Calif.). The model underwent10,000 iteration molecular mechanics minimization with the programDiscover (Accelrys, San Diego, Calif.) after its construction. Thedocking of compound 6 to this structure was performed as describedabove.

Immunoprecipitation and Immunoblotting.

Following stimulation with bFGF, VEGFA or PDGF-BB, cells were washed 1×with ice-cold PBS and lysed in RIPA buffer. For the PDGFRβautophosphorylation assay in VSMCs, 500 μg of protein from lysates wasincubated with 3 μg of anti-PDGFRβ (sc-432, Santa Cruz Biotechnology)for 1 h at 4° C. and subsequently tumbled overnight with protein A/Gbeads (Pierce) for immunoprecipitation. For the MAPK assay in HUVECs,standard SDS-PAGE and immunoblotting was performed using antibodies tophospho-MEK S217/S221, phospho-MEK S298, phospho-ERK T202/Y204,phospho-C-RAF S259 and S338 (all from Cell Signaling, Danvers, Mass.),MEK1 (Santa Cruz), ERK2 (SC), and C-RAF(SC). For the PDGFRβautophosphorylation assay, antibodies to PDGFRβ (sc-339, SC) andHRP-conjugated PY-20 (sc-508, SC) were used to detect total PDGFRβ andphospho-tyrosine levels, respectively. HRP conjugated primary orsecondary Abs were detected with SuperSignal ECL (Pierce).

Cell Viability Assays

For XTT assays, cells were grown in 96-well plates overnight and allassays were conducted in growth medium will full serum and additives.Compounds were serially diluted in DMSO and then further diluted intothe medium to give the appropriate concentration while minimizingprecipitation associated with serial diluting in medium alone.Inhibitors were added for 72 h and cell viability was quantified at 450nm after the addition of 1 mg/ml XTT solution (Sigma-Aldrich) inphenol-red free DMEM medium containing phenoxymethosulfate(Sigma-Aldrich). Dose-response curves were plotted using GraphPad Prismsoftware and EC₅₀s were calculated using this program.

Stellate Cell/Endothelial Cell Co-Culture Assay for Endothelial TubeFormation.

Early passage (p<5) human Telomerase Reverse Transcriptase-hepaticstellate cells (Stellate cells), a generous gift from Dr. David Brenner,were labeled with 10 μg/ml red fluorescent dye DiIC(3) (BD Biosciences)for 2 h at 37° C. The stellate cells were washed in PBS and mixed withearly passage HUVECs (p<5) at a ratio of 1:4 and mixed into a 3.75 mg/mlType 1 Rat tail collagen matrix (BD Biosciences) in M-199 media. 30 μlof this mixture was seeded onto each well of a half-area 96 well plate(Corning). The collagen was allowed to polymerize for 30 min at 37° C.and 100 μl of complete EBM-2 media was added to each well. Variousinhibitors were serially diluted in DMSO and subsequently added toduplicate wells 6 h post cell seeding. The endothelial tubes werestained at 24 h by adding 2 μl FITC labeled Ulex europaeus lectin(Vector labs) per well. The endothelial tubes and the stellate cellswere imaged at 48 h with a 10× objective on a confocal microscope.Images were collected across five fields per well. Tube lengths weremeasured using Metamorph software for each tube for all 10 microscopicfields from the duplicate wells. The % pericyte-covered tube length wascalculated from the ratio of tube length sums for the tubes with andwithout pericyte contact.

Mouse Matrigel™ Model of Angiogenesis

Female Nu/Nu mice were implanted with growth-factor depleted Matrigel(BD Biosciences, San Jose, Calif.) in the flank containing either PBS or200 ng of bFGF (Peprotech). The following morning mice were treated with6, control compound, or vehicle (10% HS-15 in 3.3% Dextrose) byintraperitoneal injection on a bid dosing schedule. Five days aftermatrigel implantation, mice were tail-vein injected with FITC-lectin andplugs were harvested and imaged by confocal microscopy and quantifiedafter homogenization in ice-cold PBS using a fluorescent microplatereader (Tecan Systems, Inc., San Jose, Calif.).

For immunohistochemistry, mice were implanted with matrigel plugs withthe same dosing schedule described above. On day 5, the plugs wereexcised and 5 m sections were generated. The sections were fixedaccording to the Apoptag protocol in which immunostaining was addedduring the TUNEL staining process as well as TOPRO3 (Invitrogen,Carlsbad, Calif.) for a nuclear counterstain. The sections were labeledwith anti phospho-ERK T202/Y204 (Cell Signaling) and an endothelial cellmix consisting of anti-CD31, anti-Flk, anti-VE-Cadherin (all from BDBiosciences, San Jose, Calif.), and anti-endoglin (Millipore). Primaryantibodies were added at 1:100 dilutions and the corresponding secondaryanti-Rat Ab was added at a 1:250 dilution. The tri-color images weregenerated with confocal microscopy.

Orthotopic Pancreatic Carcinoma Model

Orthotopic human pancreatic cancer xenografts from the pancreatic cancercell line XPA1-RFP were established in nude mice by surgical orthotopicimplantation (SOI). Briefly, subcutaneous XPA1-RFP tumors in theexponential growth phase were harvested and sectioned into 1 mm³ piecesin serum-free RPMI medium. Mice were anesthetized using 50% ketamine,38% xylazine and 12% agepromazine maleate injected intramuscularly at adose of 2 μl/g, and their abdominal wall was sterilized with alcohol. Asmall incision was then made in the right pararectal line through theskin and peritoneum. The body of the pancreas was exposed and a 1 mm³tumor fragment was sutured to the pancreas using a single 8-0 nylonsurgical suture (Davis-Geck, Inc. Manati, Puerto Rico). The pancreas wasthen returned to the abdomen and the peritoneum and skin were closed intwo layers using 6-0 vicryl surgical suture.

At day 3 after SOI, the animals began treatment with twice-daily IPinjections of either compound, 6 (50 mg/kg dose in 200 μl total volume),or vehicle control at 12 hour intervals. The animals were weighed andimaged every 3 days starting at day 3 post-SOI using the Olympus OV100Small Animal Imaging System (Olympus Corp. Tokyo, Japan), containing anMT-20 light source (Olympus Biosystems Planegg, Germany) and DP70 CCDcamera (Olympus Corp. Tokyo, Japan). All images were processed forcontrast and brightness with the use of Photoshop element −4. Allmeasurements were made using Image-J software. At day 12 post-SOI theanimals were sacrificed and their tumors were excised, weighed, andmeasured in 3 dimensions in order to calculate tumor volume. The tumorvolume was calculated with the formula: volume=height×width×length×0.52.Ex-vivo tumors were then compressed and imaged using the OV100. Thetotal vessel length for each tumor was measured using Image-J softwareand converted to vessel density by dividing total tumor vessel length bytumor volume.

Orthotopic Renal Cell Carcinoma Model.

Male Nu/Nu mice were anesthetized with an intraperitoneal injection of50 mg/kg ketamine and 10 mg/kg medetomidine. A small incision was madealong the left flank of the mice and the kidney was exposed. A 27-gaugeinsulin syringe was used for orthotopic injection of the SN12C-RFPcells. One million tumor cells in 20 μl of a 1:1 PBS:Matrigel mixturewere injected into the lower pole of the kidney just below the renalsubcapsule. The needle was removed after a visible blister formed andleakage of the tumor cell suspension was minimal. The abdominal wall wasclosed by suturing the peritoneal membrane followed by stapling thedermal layer. Animals which formed visible blisters upon injection inthe subcapsule with minimal leakage were used for the efficacy study.Male Nu/Nu mice with orthotopic injections of the SN12C-RFP cells wereimaged using the Olympus OV100 Small Animal Imaging System and groupedon day 7 based on weight and imaging results. The mice were dosed orallyat 100 mg/kg daily with 6 in 10% HS-15 and 3.3% dextrose. At the end ofthe study, the kidney tumors were imaged with the OV-100 system for RFPexpression and then the kidneys were resected and weighed. Thedifference in weight between the tumor bearing kidney and the normalkidney was reported.

Synthesis and Characterization of New Compounds General SyntheticMethods

Unless otherwise noted, materials were obtained from commercialsuppliers and were used without purification. Removal of solvent invacuo refers to distillation using a Bichi R-200 rotary evaporator and aWelch 2025 vacuum pump. All microwave irradiation experiments werecarried out in Initiator™ (Biotage) microwave apparatus in the standardconfiguration including the proprietary Biotage software. The reactionswere carried out in 5 mL Emrys™ microwave vials. After the irradiationperiod, the reaction vessel was cooled rapidly (1-2 min) to ambienttemperature by gas jet cooling.

¹H and ¹³C NMR spectra were recorded on Bruker AMX-II (500 MHz)spectrometer. Proton resonances are reported in parts per million (ppm)downfield from tetramethylsilane (TMS). ¹H NMR data are reported asmultiplicity (s singlet, d doublet, t triplet, q quartet, dd doublet ofdoublets, dt doublet of triplets, br s broad singlet), coupling constantin Hertz and number of protons. The ¹H and ¹³C NMR spectra were obtainedin DMSO-d₆ (99.96 DMSO-d₆ with 0.03 v/v TMS). The ¹H NMR spectra for1,2,4-triazoles were recorded at 80° C. to induce the rapid exchange ofthe N-proton of the triazole ring. Due to the intermediate-to-slowexchange of the N-proton of the triazole ring at room temperature thebroadening of the peaks is observed in both ¹H and ¹³C NMR spectrarecorded at ambient temperature. All data were processed with Xwin-NMRsoftware.

High pressure liquid chromatography was achieved using Gilson Unipoint215 Liquid Handler with 151 UV/Vis detector and Waters PrepLC™ 40 mmModule with SymmetryShield™ RP18 7 um 40×100 mm PrepPak® cartridges.Purification of compounds by high pressure liquid chromatography wasperformed at 40 mL/min flow rate with a gradient from 10% of solvent B(acetonitrile with 0.1% TFA) in solvent A (water with 0.1% TFA) to 100%solvent B in 25 min, followed by 5 min elution with 100% solvent B.

Analytical thin-layer chromatography (TLC) was performed on commercialsilica plates (EMD Chemicals, Silica gel 60 F254, 0.25 mm thickness).Compounds were visualized by UV light (254 nm). Flash chromatography wasperformed either by CombiFlash® (Companion®, Teledyne ISCO) or usingsilica gel (ICN silica 32-63 D 60 Å). Waters 2795 LC/MS with Micromass®ZQ™ and 2996 PDA detector were used to monitor the progress of reactionsand check the purity of products. Mass spectra were obtained inelectrospray ionization (ESI) positive mode. Accurate masses weremeasured by electrospray ionization (ESI) time-of-flight (TOF)reflectron experiments performed on an Agilent ESI-TOF massspectrometer. Samples were electrosprayed into the TOF reflectronanalyzer at an ESI voltage of 4000 V and a flow rate of 200microliters/minute.

In Vitro Kinase Screen and Competitive Binding Assay.

Sorafenib as well as test compounds were submitted to Invitrogen andscreened using their SelectScreen™ Profiling Service. For thecompetitive binding assay, test compounds were screened at 10 μM against70 diverse kinase targets for competitive binding using the KINOMEscan™technology (Ambit Biosciences) (Karaman, et al. (2008) Naturebiotechnology 26:127-132). 11-point Kd curves were determined for hitsidentified during the initial screen.

Cell Culture and Cell Based Screening.

HUVECs and VSMCs (Lonza, Basel, Switzerland) were maintained asrecommended with all experiments conducted at passage <6. XPA1-RFP andSN12C-RFP cells were maintained under standard culture conditions inRPMI supplemented with 10% FBS. XPA-1 cells were a gift from Dr. AnirbanMaitra at Johns Hopkins University and transduced with retrovirus toestablish stable RFP-expressing cells as described previously (Tsuji, etal. (2006) Jop 7:193-199). For cell-based assays, sub-confluent cellswere starved overnight in serum-free medium, pretreated with inhibitorand stimulated with growth factor as described in the Figure Legend. Adescription of the immunoprecipitation, immunoblotting, and antibodiesis disclosed herein.

Zebrafish Studies.

Transgenic Tg(fli1:EGFP) zebrafish embryos were purchased fromwww.zfin.org and reported elsewhere (Lawson, et al. (2002) Dev Biol248:307-318) and maintained according to (“Zebrafish—A PracticalApproach”, Oxford University Press, 2002). Compounds in DMSO stocksolutions were diluted directly into the water and vessels were imagedfor GFP expression using a Nikon cl-si confocal microscope. Apoptosiswas measured by TUNEL staining using the Apoptag in situ RedFluorescence Kit (Chemicon) with a protocol described previously(Stanton, et al. (2006) The Journal of biological chemistry281:28782-28793).

Stellate Cell/Endothelial Cell Tube Formation Assay and Mouse MatrigelModel.

This assay was performed as described in the SI Methods. The stellatecells were a gift from Dr. David Brenner and previously described(Schnabl, et al. (2002) Laboratory investigation; a journal of technicalmethods and pathology 82:323-333). The 3D-collagen assay for tubeformation was performed as previously described (Koh, et al. (2008)Methods in enzymology 443:83-101). The mouse matrigel model wasperformed as previously described (Eliceiri, et al. (1999) Molecularcell 4:915-924 5) and further details of immunohistochemistry and animaldosing can be found herein.

Orthotopic Pancreatic Carcinoma and Renal Cell Carcinoma Models.

Six-week old male Nestin-GFP nude mice described previously (Amoh, etal. (2005) Cancer research 65:5352-5357; Amoh, et al. (2006) J Surg Res132:164-169) underwent surgical orthotopic implantation of XPA-1-RFPpancreatic tumor cells as described previously (Katz, et al. (2003)Cancer research 63:5521-5525, 39; Bouvet, et al. (2002) Cancer research62:1534-1540) and in further detail disclosed herein. The renal cellcarcinoma model was used as previously described (An, et al. (1999) ClinExp Metastasis 17:265-270) and is described in further detail herein.

Statistical Calculations.

All statistical evaluation was done using NCSS 2007 statistical software(Kaysville, Utah). For comparisons between two groups a two-tailedt-test was used for data with equal variance and the Aspin-Welch UnequalVariance Test was used for data with unequal variance. For comparisonbetween more than two groups a One-way ANOVA was used. P-values arenoted in the figure legends.

Example 1 Preparation of Triazole Compound 6

Compound 6 was prepared from commercially available4,6-dichloro-2-(methylthio)pyrimidine followed scheme 4. To preparecompound 6.1, 4,6-Dichloro-2-(methylthio)pyrimidine was reacted withammonia in THF in a sealed tube to provide4-Amino-6-chloro-2-(methylthio)pyrimidine in good yield. Amino group ofthe resulting compound was protected with suitable protecting group suchas BOC to afford compound 6.1. Compound 6.1 was then reacted with methyl4-hydroxybenzoate in the presence of base to provide the amine protectedintermediate, 2 which upon deprotection under acidic condition to affordmethyl 4-(6-amino-2-(methylthio)pyrimidin-4-yloxy)benzoate (3). Reactionof compound 3 with hydrazine hydrate in methanol provided4-(6-amino-2-(methylthio)pyrimidin-4-yloxy)benzohydrazide (6.4) in goodyield. The hydrazide was then reacted with S-methylN-[3-(trifluoromethyl-phenyl)thiourea in the presence of base to yieldcompound 6.

Example 1-1 Preparation of 4-Amino-6-chloro-2-(methylthio)pyrimidine

Reagents:

Raw materials Quantity M. Moles Eq. 4,6-dichloro-2-(methyl- 400 g 1952.05 1.0 thio)pyrimidine Ammonia in THF 4.0 L — — 10 Vol (16% Assay)Hexane Lot-1 400 mL — — 2 Vol Hexane Lot-2 400 mL — — 2 Vol Water 2 ×400 mL — — 2 Vol

Experimental Procedure:

Take ammonia in THF into a 2 L autoclave and add 4,6-dichloro-2-(Methylthio) pyrimidine slowly.

Heat the reaction mixture to 50-60° C. and maintain the reaction at50-60° C. for 3-4 hours (Inbuilt pressure 7-8 Kg/cm²).

Check the progress of the reaction by TLC. Upon completion, the reactionwas brought to 25-35° C.

Concentrate the reaction mixture under vacuum.

Charge Hexane and stir for 30-45 minutes at 25-35° C.

Filter the solid and wash the solid with Hexane.

Wash the solid with water (2×400 mL).

Dry the solid at 25-35° C. till M.C reaches to less than 2%.

Yield 352.0 g % of Yield: 97.77%. Purity by HPLC: 99.07%.

Other suitable conditions such as ammonia in MeOH or dioxane could beused accordingly when different analogs are used.

Example 1-2 Preparation of 4-Amino-6-chloro-2-(methylthio)pyrimidine,6.1

Reagents:

Raw materials Quantity M.W. Moles Equiv 4-Amino-6-chloro-2- 300 g 175.51.7 1.0 (methylthio)pyrimidine THF 1.5 L — — 5 Vol K₂CO₃ 519.76 g 138.213.7 2.2 Boc anhydride 31.0 g 218 4.2 2.5 DMAP 20.85 g 122  0.17 0.1Ethyl acetate Lot-1 900 mL — — 3 Vol Water 750 mL — — — Saturated Sodium900 mL — — 3 Vol chloride solution Sodium sulphate 50 g — — — Ethylacetate Lot-2 300 mL — — —

Experimental Procedure

Charge 4-Amino-6-chloro-2-(methylthio)pyrimidine and THF into a cleanand dry round bottom flask at 25-35° C.

Add K₂CO₃ lot wise at 25-35° C. in 5-10 minutes.

Add Boc anhydride slowly at 25-35° C. in 15-30 minutes.

Stir at 25-35° C. for 15-30 minutes.

Add DMAP slowly in 5-10 minutes at 25-35° C.

Maintain the reaction mass at 25-35° C. for 1-2 hours.

Check the progress of reaction by TLC.

Upon completion, charge ethyl acetate and water.

Stir the reaction mass at 25-35° C. for 10-15 minutes.

Separate the aqueous layer and organic layer.

Wash the organic layer with saturated sodium chloride solution.

Dry the organic layer over sodium sulphate

Filter sodium sulphate out

Wash the sodium sulphate with Ethyl acetate.

Concentrate the organic layer completely under vacuum.

Yield: 632 g

% of Yield: 98.46.

Purity by HPLC: 77.98% Di-Boc compound.

11.49% Mono Boc compound.

Alternatively, compound 6.1 can be prepared by addition of (BOC)₂NHanion to the corresponding 4,6-dichloropyrimidine or other suitablemethods known in the art.

Example 1-3 Preparation of Compound 2

Reagents:

Raw materials Quantity M. Wt Moles Equiv Compound 6.1 650 g 375.5  1.71.0 DMF 1.95 L — — — Methyl hydroxyl benzoate 266.42 152.15 1.9  1.011,4-diazabicyclo[2.2.2]octane 235 mL 112.18 2.0 1.2 Water Lot-1 6.5 L —— 10 Vol Water Lot-2 1.3 L — — —

Experimental Procedures:

Charge compound 6.1 into a clean and dry RB flask at 25-35° C.

Charge DMF into RB flask at 25-35° C.

Charge Methyl hydroxyl benzoate at 25-35° C.

Charge 1, 4-diazabicyclo[2.2.2]octane (DABCO) into RB Flask at 25-35° C.

Stir the reaction mass at 25-35° C.

Check the progress of the reaction by TLC.

IF TLC complies add water Lot-1.

Stir at 25-35° C. for 1-2 hours.

Filter the solid and wash the solid with water Lot-2.

Dry the solid at 25-35° C. M.C reaches to below 3%.

Yield: 680 g

% of Yield: 80%.

Purity by HPLC: 90.56%.

It was found that other suitable amine bases, preferably a tertiaryamine base (e.g. DBU, pyridine, DMAP) can be used to achieve good yield.For example, pyridine has been used at reflux for 18 h to give 88% yieldof product. THE or acetonitrile were used as alternative solvents toDMF, with acetonitrile preferred.

Example 1-4 Preparation of Compound 6.3

Reagents

Raw materials Quantity M. Wt Moles Equiv Compound 6.2 300 g 491.3 0.611.0 DCM 278 mL — — — Trifluoroacetic acid 278 mL 114   3.66 6.0Diisopropylether Lot-1 600 mL — — — Diisopropylether Lot-2 300 mL — — —Water Lot-1 3.0 L — — 10 Vol Water Lot-2 600 mL — — — Sat. NaHCO₃Solution ~500 mL — — —

Experimental Procedures

Charge compound 6.2 into a clean and dry RB flask at 25-35° C.

Charge DCM into RB flask at 25-35° C.

Cool the reaction mass to 0-5° C.

Add TFA at 0-5° C. slowly (or HCl in dioxane).

Raise the reaction mass temperature to 25-35° C.

Stir the reaction mass at 25-35° C. for 18-20 hours.

Check the proceeding of reaction by TLC.

If TLC complies distill the reaction mass under vacuum below 40° C.

Charge Diisopropyl ether at 25-35° C.

Stir at 25-35° C. for 30-45 minutes.

Filter the solid and wash the solid with Diisopropyl ether Lot-2.

Suck dry the solid for 15 minutes.

Charge the wet solid into a clean and dry beaker.

Charge Water Lot-1 at 25-35° C.

Slowly adjust the reaction mass pH to 9-10 with sat.NaHCO₃ solution.

Stir the reaction mass at 25-35° C. for 15-30 minutes.

Filter the solid and wash the solid with water Lot-2.

Dry the solid at 25-35° C. till to get constant weight.

Yield: 160 g.

% of Yield: 88%.

Purity by HPLC: 98.68%.

It was also found that other suitable conditions such as HCl/dioxane,HCl/MeOH, etc could be employed to remove the Boc protecting groups.

Example 1-5 Preparation of Compound 6.4

Reagents

Raw materials Quantity M. Wt Moles Equiv Compound 6.3 250 g 291.3 0.81.0 Methanol 1.25 L — — 5 Vol Hydrazine hydrate 257.46 mL 50  5.1 6.0Water Lot-1 1000 mL — — — Water Lot-2 500 mL — — —

Experimental Procedures

Charge compound 6.3 into a clean and dry RB flask at 25-35° C.

Charge Methanol and Hydrazine hydrate (or anhydrous hydrazine) at 25-35°C.

Heat the reaction mass to reflux.

Stir at reflux for 9-10 hours.

Check the progress of by TLC.

Upon completion, distill off solvent completely under vacuum at 40-50°C.

Charge Water Lot-1 at 25-35° C.

Stir at 25-35° C. for 1 hour.

Filter the solid and wash the solid with water Lot-2.

Dry the solid at 25-35° C. till to get constant weight.

Yield: 191 g.

% of Yield: 77%.

Purity by HPLC: 99.54%.

Example 1-6 Preparation of Compound 6

Reagents

Raw materials Quantity M. Wt Moles Equiv Trifluoromethyl phenyl 110 g220 0.49 1.0 thiourea 6.5 DCM 1.1 L — — 10 Methyl iodide 212.79 141.91.49 3.0 Compound 6.4 120 g 291.3 0.41 0.83 Acetonitrile 600 mL — — —2,6 lutidine 220.4 g 107 2.05 4.0 Ethyl acetate Lot-1 1.2 L — — 10 WaterLot-1 1.2 L — — 10 10% Citric acid solution 1.2 L — — 10 Water Lot-2 1.2L — — 10 Na₂SO₄ 50 g — — — Ethyl acetate Lot-II 220 mL — — 2 Vol

Experimental Procedures

Charge trifluoro methyl phenyl thiourea (6.5) into a clean and dry RBflask at 25-35° C.

Charge DCM and Methyl iodide at 25-35° C.

Heat the reaction mass to reflux.

Stir at reflux for 3-4 hours.

Check the progress of by TLC.

Upon completion of the reaction, distill off solvent completely undervacuum at 40-50° C.

Charge acetonitrile to above crude material at 25-35° C. at N₂atmosphere.

Charge compound 6.4 and 2,6-lutidine.

Heat the reaction mass to reflux.

Maintain the reaction mass under reflux for 16-17 hours at N₂atmosphere.

Check the progress of the reaction by TLC.

Upon completion, distill off the solvent completely under vacuum.

Dissolve the residue in Ethyl acetate Lot-1.

Wash the Ethyl acetate layer with Water Lot-1.

Wash the Ethyl acetate layer with 10% Citric acid solution.

Wash the Ethyl acetate layer with Water Lat-2.

Dry the Ethyl acetate layer over Sodium sulphate.

Wash the sodium sulphate with Ethyl acetate Lot-2.

Distill off the total Ethyl acetate layer under vacuum at 50-55° C.

Filter the solid and wash the solid with water Lot-2.

Dry the solid at 25-35° C. till to get constant weight.

Yield: 245 g. (Crude)

Purity by HPLC: 86.4%.

It was found that the use of suitable base would greatly improve theyield and purity. For example, the use of Cs₂CO₃ in DMF failed to affordproduct and the use of pyridine (5 eq) as solvent/base at reflux for 5 hafforded 55% yield, contaminated with isosteric oxadiazole analog whichwas difficult to remove. Conditions developed herein with lutidinesuppress the formation of oxadiazole analog and provide efficient andeconomic ways to prepare compounds of interests. Other suitable solventsfor the reaction may be acetonitrile, DMF, N-methyl-pyrrolid-2-one(NMP), ^(t)BuOH or ^(i)PrOH.

Example 2 Mesylate Salt of Compound 6

The Mesylate salt was prepared from either crude or purified compound 6with about same yield. The analytical data shows bis-mesylate salt withmonohydrate solvate.

Reagents

Raw materials Quantity M. Wt Moles Equiv crude compound 6 39 g 459 0.081.0 Methane sulfonic acid 16.31 g 96 0.16 2.0 Methanol Lot-1 160 mL — —— Chilled Methanol Lot-2 50 mL — — — Methanol Lot-3 200 mL — — — ChilledMethanol Lot-4 60 mL — — —

Procedures

Charge crude compound 6 into a clean and dry RB flask at 25-35° C.

Charge Methanol Lot-1 into RB flask at 25-35° C.

Stir at 25-35° C. for 10-15 minutes.

Add Methanesulfonic acid slowly at 25-35° C.

Stir the reaction mass at 25-35° C. for 3-4 hours.

Filter the solid and wash the solid with Chilled methanol Lot-2(Precooled).

Dry the solid at 25-35° C. for till to get constant weight. (Purity byHPLC: 94.5%)

Charge the solid into a clean and dry RB flask at 25-35° C.

Charge Methanol Lot-3 at 25-35° C.

Stir at 25-35° C. for 1-2 hours at 25-35° C.

Filter the solid and wash the solid with chilled methanol Lot-4(Precooled).

Dry the sold at 25-35° C. till to get constant weight.

Yield: 38 g

Purity by HPLC: 98.2%.

Mesylate content: 33.28%(Bis Mesylate)

Moisture content: 6.08%(Mono hydrate)

Example 3 Preparation of Pyridine Hydrazide Precursor 44

Compound 42 is prepared from methyl 3-mercaptobenzoate (41) and compound42 under basic condition (e.g. 2-6-lutidine or DABCO). The esterfunctional group of compound 43 is then converted to hydrazide first(hydrazine reaction) and then Boc protecting group is deprotected underacidic condition (e.g. TFA) to afford compound 44.

Example 4 Preparation of Quinazoline Hydrazide 48

Compound 47 is prepared from methyl 4-(benzyloxycarbonylamino)benzoate(45) and compound 46 under basic condition (e.g. 2-6-lutidine orpyridine). The ester functional group of compound 47 is then convertedto hydrazide first (with hydrazine) and then Cbz protecting group isdeprotected under hydrogenation condition to afford compound 48.

Example 5 Preparation of Triazole 49

Triazole compound 49 is prepared under similar condition as described inExample 6 from compound 48.

Example 6 Design of a Selective Type II PDGFRβ/B-RAF Inhibitor

While the overall homology of the PDGFRβ and B-RAF kinase domains isrelatively low (29.1% homology and 47.3% similarity, see FIG. 6A), thesetwo kinases feature structurally related type II pockets, guiding thedesign of an amino-triazole scaffold to fit into the allosteric site inthe inactive conformation of these and other kinases while avoiding theATP pocket (FIG. 1A). Amino-triazole based compounds were screened inhuman primary cell-based assays for their ability to suppressPDGF-BB-mediated PDGFRβ autophosphorylation in vascular smooth musclecells (VSMCs) and growth factor-mediated MEK and ERK phosphorylation inendothelial cells (ECs) (FIGS. 1B and C). Structure-activityrelationships demonstrate the critical substituents for cell-basedPDGFRβ and RAF inhibition (FIG. 1D) and are further described herein.Active compounds were then screened for anti-angiogenic activity in thedeveloping zebrafish (from 20-48 hours post fertilization (hpf)) byevaluating the growth of intersegmental vessels (FIG. 3). Successiverounds of molecular modeling, chemical synthesis, as well as cell-basedand zebrafish screening were performed to refine the active molecules.

Example 7 Effect of Compounds on Cell Viability

The effects of compound 3 vs. 6 on VSMC or EC viability were testedsince compound 3 inhibited PDGFRβ, while compound 6 inhibited both RAFand PDGFRβ (see SAR in FIG. 1D). Compound 6 inhibited VSMC viabilitywith an EC₅₀ of 0.59 μM, whereas 3 produced an EC₅₀ of 15.0 μM (FIG.1E). Imatinib (PDGFRβ among its kinase targets) did not demonstrate anyinhibition of VSMC viability at the highest concentration tested (20μM). Compound 6 inhibited endothelial cell viability with an EC₅₀ of0.54 μM, whereas 3 produced an EC₅₀ of 18.04 μM (FIG. 1F) confirming ourSAR demonstrating that the methylthiol group and both PDGFRβ and p-MEKinhibitory activity are critical for the cytotoxic effects observed.Interestingly, sorafenib, a RAF inhibitor with both type II and ATPcompetitive binding properties, produced an EC₅₀ of 13.24 μM on theviability of the ECs indicating that sorafenib was 25-fold less potentthan 6 in this cell-based assay.

Example 8 Comparison of Compound 6 to Sorafenib In Vitro

Compound 6 and sorafenib were further compared in a panel of in vitroATP-dependent kinase assays consisting of several targets that areinhibited by sorafenib. Compound 6 did not inhibit any of the followingactive kinases: B-RAF, C-RAF, VEGFR1, VEGFR2, Flt3, Kit, and PDGFRβ aswell as several others, even at 10 μM (Table 1). This is not surprisinggiven that compound 6 requires the inactive conformation of the enzymefor interaction. In contrast, sorafenib inhibits the kinase activity ofB- and C-RAF in addition to VEGFR2 and several other receptor tyrosinekinases whereas compound 3, like 6, did not inhibit any of the activekinases tested.

TABLE 1 In vitro kinase panel comparing compounds 3, 6, and sorafenib %Inhibition at 10 μM Kinase Target Cmp 3 Cmp 6 sorafenib ABL1 4 3 50 AKT11 2 2 BRAF 17 −1 84 CDK2 2 2 2 CSF1R 79 23 101 CK2 alpha 1 −3 0 −3 EGFR−2 −3 −3 EGFR L858R −3 −10 −5 EGFR L861Q −1 −4 −4 FGFR1 9 6 44 FGFR2 −1−2 83 FGFR3 −12 −13 37 FGFR3 K650E 4 −1 78 FGFR4 −6 −7 7 FLT1 (VEGFR1) 5−2 93 FLT3 −4 −2 9 FLT3 D835Y 29 9 72 IGF1R −4 0 −3 KDR (VEGFR2) 31 16104 KIT 74 28 37 MEK1 −5 −2 0 MEK2 −8 −6 −4 ERK2 −4 −5 0 JNK3 −8 −8 −4ERK1 1 2 3 JNK1 −5 −3 −2 JNK2 −7 −4 10 MAPKAPK2 1 2 2 MET (cMet) 7 3 2MET M1250T 4 −1 3 PAK2 2 1 2 PDGFR beta 35 20 93 PKC alpha −5 −1 −2 PKCbeta I −3 2 −1 PKC beta II −3 −4 −3 PKC delta −1 −1 −4 PKC epsilon −3 −1−2 PKC gamma 6 5 −3 PKC eta 1 3 2 PKC iota −2 0 −2 FAK2 −4 −4 −3 CRAFY340D Y341D 38 3 95

Next, compound 6 was analyzed in a competitive binding assay (Karaman MW, et al. Nature biotechnology (2008) 26:127-132) against 70 kinases(with the majority as inactive) representing diverse family members ofthe kinome (FIG. 2 and Table 2). Relative to other type II inhibitors,imatinib and sorafenib, compound 6 displays improved selectivity, whichis represented on the kinase dendrograms (FIG. 2). Among kinases,compound 6 inhibited only PDGFRα and β with Kds of 300 and 520 nM,respectively, as well as Flt3 and c-Kit at 52 and 170 nM, respectively(Table 3). A panel of CDKs were tested and weak binding was observedwith CDKL2 (5.1 μM) and CDK11 (7.5 μM). While compound 6 did not inhibitRAF in this assay, this is not surprising since the RAF construct usedin this assay has an N-terminal regulatory domain truncation likelyinfluencing the allosteric conformation.

TABLE 2 Kinases screened with 10 uM compound 6 in the KINOMEscanprofiling assay at Ambit Biosciences used to construct FIG. 2. Kinase %Control ABL1 100 AMPK-alpha1 72 ARK5 70 AXL 87 BMX 97 BRAF 91BRAF(V600E) 98 CHC2L1 70 CDC2L2 83 CDK11 32 CDK11 32 CDK2 87 CDK3 91CDK5 88 CDK7 97 CDK8 67 CDK9 100 CLK2 84 CSF1R 51 CSNK1E 47 DYRK1B 85EPHA2 100 EPHB4 94 ERK2 90 FAK 100 FGFR2 100 FLT1 81 FLT3 2.8FLT3(D835H) 17 FLT3(D835Y) 18 FLT3(ITD) 3.1 FLT3(N841I) 3.1 FLT4 100GSK3B 90 IGF1R 100 INSR 100 JNK1 98 KIT 0 KIT(D816V) 76 KIT(V559D) 0.1KIT(V559D, T670I) 56 KIT(V559D, V654A) 15 LIMK2 72 LKB1 100 MEK1 84 MET94 MLK3 96 MYO3A 94 p38-alpha 100 PAK1 100 PAK4 100 PDGFRA 0.25 PDGFRB0.5 PFTK1 71 PIK3CA 100 PIM1 96 PKAC-alpha 100 PRKCD 74 PRKCE 91 PRKD1100 RAF1 52 RET 95 RIOK3 100 RIPK1 84 SRC 90 SRPK2 100 TIE1 76 TIE2 88TRKA 64 VEGFR2 80

TABLE 3 Kinome profile of compound 6 in the competitive binding assay(KINOMEscan) Kinase Kd (nM) FLT3 52 KIT 170 PDGFRα 300 PDGFKβ 520 CDKL25,100 CDK11 7,500 ABL >10,000 B-RAF >10,000 CSF1R >10,000 FGFR2 >10,000p36α >10,000 PAK1 >10,000 RET >10,000 VEGFR2 >10,000

Although compound 6 failed to inhibit truncated RAF in the competitivebinding assay, it completely disrupted phosphorylation of ERK T202/Y204in cells expressing the constitutively active mutant of B-RAF (V600E),providing support that compound 6 directly targets RAF in cells (FIG.7B). Additionally, compound 6 did not inhibit activation of either FGFR1or VEGFR2 in endothelial cells (FIG. 7E) or in vitro (Table 2), but didinhibit the activation of PDGFRβ in vascular smooth muscle cells (FIG.1B and FIG. 7E). A summary of the phospho-sites examined in bFGF orVEGFA stimulated ECs is found in Table 4.

TABLE 4 Cell-based profiling of compound 6. Phospho-Protein InhibitionC-RAF S259 − C-RAF S621 − C-RAF S336 + MEK-1/2 S217/S221 + ERK-1/2T202/Y204 + ERK-5 T218/Y220 − Bad S112 or S155 − Src Y416 − PanPhospho-PKC − P38 T180/Y182 − SAPK T183/Y185 − FAK Y861 − Akt S473 −VEGFR2 (phospho-tyrosine) − FGFR1 (phospho-tyrosine) − PDGFRβ(phospho-tyrosine) in SMCs +

HUVECs were pretreated with compound 6 for 1 h and stimulated with bFGFfor 5 min. For PDGFRβ, VSMCs were pretreated with compound 6 for 1 h andstimulated with PDGF-BB for 7 min since HUVECs do not express thisreceptor.

Example 9 Phosphorylation Analysis of Compound 6

The specificity of compound 6 for RAF was further analyzed by evaluatingits effect on specific phosphorylation sites both within and outside theRAF activation domain. Both bFGF and VEGF lead to phosphorylation ofserine 338 (via PAK) within the activation domain of C-RAF while serine259, which mediates the coupling of C-RAF to the adaptor protein 14-3-3(Rommel C, et al. Oncogene (1996) 12:609-619), is constitutivelyphosphorylated (FIG. 7C). Compound 6 selectively blocked S338phosphorylation, yet did not influence S259 (FIG. 7C), suggesting thatits interaction with RAF preferentially influences its activationdomain. Importantly, compound 6 did not inhibit PAK since it did notblock PAK-mediated phosphorylation on MEK S298 in these cells (FIG. 1C).

Example 10 Evaluation of the Effect of Compound 6 on RAFHeterodimerization

Recent studies demonstrate the importance of B-RAF/C-RAFheterodimerization for effective MAPK signaling since heterodimerizationdramatically increases the activity of both B-RAF and C-RAF (Rushworth,et al. (2006) Mol Cell Biol 26:2262-2272; Weber, et al. (2001) CancerRes 61:3595-3598). Furthermore, certain mutations which induce an “openconformation” of B-RAF promote constitutive binding to C-RAF in cancercells and this heterodimerization activates C-RAF and MEK signaling(Garnett, et al. (2005) Molecular cell 20:963-969). This defines RAFheterodimerization as an intriguing target for disrupting RAF activityin cells.

To evaluate the effect of compound 6 on RAF heterodimerization,endogenous RAF heterodimerization was induced in ECs by stimulating thecells with bFGF or VEGF and the ability of compound 6 to impact theformation of this complex was studied (FIG. 7D). Treatment of cells withcompound 6 completely inhibited this heterodimer formation as well asERK phosphorylation in response to either growth factor, while compound3 had no effect (FIG. 7D). In addition, a MEK inhibitor, U0126,inhibited phosphorylation of ERK as expected, but greatly increased RAFheterodimerization above that achieved with growth-factor stimulationalone, suggesting the possibility of a feedback loop upon MEKinhibition. The finding that compound 6 inhibits phosphorylation of MEKand ERK in endothelial cells stimulated with bFGF or VEGF (FIG. 1C)provides evidence that compound 6 targets both B-RAF and C-RAF in cellsand inhibition of MAPK signaling by disrupting RAF heterodimerizationmay be an ideal mechanism to deal with the compensatory roles of B-RAFand C-RAF.

Example 11 Analysis of the Effects of Compound 6 on New Blood VesselGrowth During Zebrafish Embryogenesis

The effects of compound 6 were further analyzed to assess itsmechanistic impact on new blood vessel growth during zebrafishembryogenesis. In embryos treated with compound 6 (1 μM in the water)the endothelial cells migrated away from the dorsal aorta as typicallyobserved in control animals yet by 48 hpf they failed to form matureintersegmental vessels and similarly impacted the developing vasculaturewithin the head region (FIG. 3A) while compound 3, which inhibits PDGFRβbut not B-RAF, had no effect. Following treatment, the blood vesselsappeared highly disorganized and lacked the capacity to support bloodflow. In contrast, fish treated with compound 3, even up to 10 μM, wereindistinguishable from non-treated controls (FIG. 3A). It is importantto note that compound 3 has Kds of 1.4, 3.7, 32, and 40 nM against Flt3,KIT, PDGFRα and PDGFRβ, respectively, in the competitive binding assayused in FIG. 2. Since compound 3 is ineffective at inhibitingangiogenesis in the zebrafish developmental embryogenesis model, it isclear that Flt3 and KIT do not play a role in blood vessel formation.Interestingly, SU5416 (Semaxanib)(12), a VEGFR2 inhibitor, whilecompletely disrupting endothelial cell migration and neovascularizationin the tail, had minimal effects on ocular vessels in these animals,suggesting that VEGF does not play an important role during ocularvascular development.

Example 12 Analysis of the Temporal Effects of Compound 6 onAngiogenesis

To analyze the temporal effects of compound 6 on angiogenesis, zebrafishwere treated with compound 6 at 18 hpf and then analyzed at 35 hpf aslumens were beginning to form. Compound 6 showed no apparent effect onthe vessels at 35 hpf, whereas imaging at 48 hpf suggests compound 6impacts a late step in lumen formation (FIG. 3B). This was followed bythe induction of apoptosis as shown by TUNEL stained intersegmental ECsat 48 hpf (FIG. 3B). In contrast, addition of compound 6 at 30 hpf(atthe time when intersegmental vessel structure is first established) didnot suppress the vascular growth and patterning (FIG. 3C) suggestingthat once lumen formation is initiated, neovessels in these animals areresistant to the effect of 6. In contrast, sorafenib not only preventedthe formation of new blood vessels but it also disrupted matureintersegmental blood vessels when added at 30 hpf (FIG. 3C).Additionally, treatment of embryos with compound 6 at 48-72 hpf had noeffect on mature intersegmental vessels and the zebrafish were allviable, whereas treatment with sorafenib at this late time point induceddeath in all animals tested (FIG. 3D). Thus, compound 6 appears todisrupt a late step in neovascularization without detectable toxicitywhile sorafenib suppresses multiple processes during embryogenesisleading to lethality.

Example 13 Analysis of Anti-Angiogenic Effect by Dual Inhibition ofPDGFRβ and RAF

The requirement for dual inhibition of PDGFRβ and RAF for angiogenesisinhibition was investigated in the zebrafish model. Only the combinationof RAF inhibition (GW 5074) and PDGFRβ inhibition (imatinib) led to asimilar phenotype to compound 6, in which angioblasts migrate from thedorsal aorta and posterior cardinal vein but fail to form functionalvessels with open lumens capable of supporting blood flow (FIG. 4A).Representative images of the Tg:fli1-EGFP zebrafish embryos demonstratethat 5 μM imatinib or 1 μM GW 5074 do not have an effect onintersegmental vessel formation and functional vessels with open lumensare observed (FIG. 4A).

Example 14 Co-Culture Angiogenesis Assay

To validate the findings of Example 10, a co-culture angiogenesis assaywas utilized to evaluate similar combinations of PDGFR and RAFinhibitors. In the assay, HUVECs are mixed with hepatic stellate cells(a pericyte found in the perisinusoidal space of the liver) in a3D-collagen gel. The co-culture produced endothelial tubes with pericytecontacts (from the stellate cells) that can be imaged and quantified asshown in FIGS. 4B and C. Treatment of the co-cultures with 2.5 μM 6 ledto a dramatic reduction in both overall endothelial tube formation aswell as % pericyte-covered tube length (FIGS. 4B and C). Addition of 2.5μM 3 or 1.0 μM imatinib (Im), which are both PDGFRβ inhibitors, did notaffect endothelial tube formation or cause a significant change inpericyte coverage of the endothelial tubes (FIGS. 4B and C). Apreviously identified RAF inhibitor, GW 5074 (13), did not affectpericyte coverage or tube length alone, but combination with 1.0 μMimatinib produced a similar decrease in the % pericyte-covered tubelength compared to 6 (FIGS. 4B and C).

Example 15 Assessment of Anti-Angiogenic Properties of Compound 6 in aMammalian Model

To assess the anti-angiogenic properties of compound 6 in a mammalianmodel, mice were subcutaneously injected with Matrigel containing bFGFto induce neovascularization and systemically treated with compound 6 at50 mg/kg, ip, bid (pharmacokinetic analysis of the dose and formulationof compound 6 used indicated a C_(max) of 3.6 μg/ml or 7.7 μM, T_(1/2)corresponding to 11.5 h, and an AUC^(0-12h) of 14.7 μg*h/ml). At thisdose, compound 6 completely blocked angiogenesis relative to vehiclecontrol (FIG. 9A) To monitor the effects of compound 6 on RAF signalingin vivo, cryosections of bFGF stimulated tissues for the presence ofp-ERK immunostaining was evaluated. bFGF stimulation of these tissuesled to intense p-ERK staining in both invasive endothelial and stromalcells. Systemic treatment of animals with compound 6 blocked MAPKpathway signaling within endothelial cells as suppression in the p-ERKstaining in these cells was observed (FIG. 9B). Additionally,vehicle-treated mice displayed intense p-PDGFRβ Y751 in SMA-positivecells (FIG. 9C), while mice treated with compound 6 demonstrated acomplete suppression in the p-PDGFRβ Y751 signal associated with thestromal compartment surrounding endothelial cells (FIG. 9C). IntenseTUNEL staining among the neovessels in these tissues was observed butmuch less staining associated with the stromal cells adjacent to thesevessels (FIG. 9D). Compound 6 blocked angiogenesis and inhibitedRAF/PDGFRb in mice. Therefore, compound 6 disrupts a survival signal inactively growing blood vessels.

Example 16 Effects of Compound 6 for Preventing Tumor Growth in anOrthotopic Pancreatic Carcinoma Model

RFP expressing XPA-1 pancreatic tumor xenografts were implanted into thepancreas of Nestin GFP-mice enabling detection of both developing bloodvessels (GFP) and primary tumor (RFP). Animals were systemically treatedwith either vehicle or compound 6 (50 mg/kg, bid) beginning 3 days aftersurgical orthotopic implantation (SOI) of a tumor fragment. Tumor growthwas monitored non-invasively by whole body imaging using the OlympusOV100 Small Animal Imaging System to detect the RFP signal from thetumor implant within the pancreas. Tumor-growth was completelysuppressed in animals treated with compound 6 compared to the vehiclealone 12 days post SOI. Representative time course images (lateral view)from three animals demonstrate that the growth of pancreatic tumorstreated with compound 6 was suppressed and RFP intensity was abolishedby day 12 after SOI compared to vehicle treated animals (FIG. 5A). Theplot of tumor surface area over time reveals the increase in the vehicletreated animals relative to the animals treated with 6 (FIG. 5B).Clearly, 6 suppressed tumor growth in this model but caused no weightloss (FIG. 5C) or detectable toxicity (data not shown).

To precisely measure the effects of compound 6 on tumor growth, thetumors were resected and weighed on day 15. Animals treated with 6produced an average tumor weight of 26.7 mg compared to 74.1 mg forvehicle treated animals (FIG. 5D). At this time, the GFP-labeled bloodvessels were imaged and the tumor associated blood vessel density wasquantified by measuring the ratio of total blood vessel length to tumorvolume (FIGS. 5E and 5F). Tumors treated with 6 were substantially lessvascularized relative to vehicle treatment and images of the GFP-labeledtumor vasculature showed a significant reduction in the total bloodvessels present (FIG. 5E). The mean vessel length/tumor volume was 2.5mm/mm³ compared to 0.2 mm/mm³ for vehicle and 6, respectively (FIG. 5F).Theses results indicate that compound 6 prevents tumor growth in anorthotopic pancreatic carcinoma model.

Example 17 Effects of Compound 6 on Tumor Growth after OralAdministration

To test the effects of 6 on tumor growth after oral administration,human SN12C renal cells expressing RFP were injected into the kidneycapsule of nude mice and tumors were allowed to develop for 7 days.Compound 6 was dosed at 100 mg/kg and demonstrated favorablepharmacokinetics with a C_(max) of 4.9 μg/ml, T_(1/2) of 6.1 h, and anAUC^(0-24h) of 8.7 μg*h/ml. Suppression of tumor growth was readilyobserved in those animals treated with 6 (FIG. 10A). On day 26, thekidneys were excised from these animals and the weight due to tumor wascalculated by subtracting the weight of the normal kidney from theweight of the tumor-bearing kidney for each animal. The average tumorburden of the vehicle group was 132±39 mg compared to 49±18 mg for thecompound 6 treated group (FIG. 10B). This demonstrates the oral activityof 6 in preventing tumor growth of an orthotopic renal cell carcinomamodel.

Example 18 Quantification of the Compound 6 Effect on the ISV Volume

Representative views of zebrafish embryos treated as in FIG. 3A (imagesrepresent merged phase and GFP fluorescence views of the head and trunkregions of Tg:fli1-EGFP embryos). Scale bar=200 μm. (FIG. 8C)Quantification of ISV volume from embryos treated as in FIG. 3A. (FIG.8D) Quantification of ISV volume from embryos treated as in FIG. 3B. Tomeasure the intersegmental vessel (ISV) volume, individual ISVs weredigitally isolated using the Imaris countersurface/isosurface functions.30 independent ISVs from 4 independent embryos were used for themeasurement. Reported +/−sem.

Example 19 Effects of Compound 6 Regarding Intimal Hyperplasia in Mice

Restenosis is a process that commonly results from balloon angioplastyand/or stent placement resulting in eventual occlusion of arteries by aprocess described as neointimal hyperplasia (NIH). After arterialinjury, an over-proliferation of vascular smooth muscle cells occurswhich has previously been shown to be dependent on both PDGFRα/β(Englesbe, et al. (2004) J Vasc Surg 39, 440-6) and MAPK pathwayactivation (Li, et al. (2005) Circulation 111, 1672-8; Pintucci, et al.(2006) Faseb J 20, 398-400). Therefore, the combination of PDGFRβ/B-RAFinhibition would be an ideal treatment for NIH. As shown in FIG. 1E,compound 6 potently inhibited VSMC viability in the presence of serumand was therefore tested in a mouse model of arterial wire-injury. Mousecarotid arteries were wire-injured and treated the following day with100 mg/kg, qd, via oral gavage. Representative sections of the injuredarteries, stained with hematoxylin and eosin (FIG. 11A), were used tocalculate the intimal/medial ratio—a measurement of the thickness of theinner two layers of the artery wall, which correlates with the size ofthe vessel lumen. Dosing with compound 6 one day after artery injurydrastically reduced the intimal/medial ratio (FIG. 11B). As expected bythe reduction in the intimal/medial ratio, the percent stenosis isgreatly reduced by treatment of compound 6 relative to vehicle treatment(FIG. 11C). As a pharmacodynamic endpoint, representative arteries wereexcised 4 h after the final dose and lysates were immunoblotted todetect activated PDGFRβ. Treatment of the mice with 6 inhibited PDGFRβactivation in these tissues when compared to vehicle treated mice (FIG.11D), confirming inhibition of the target in vivo following oraladministration.

Example 20 Effects of Compound 6 for Inhibiting Tumor Cell Viability

Compound 6 was shown to have broad significant tumor cell killing orgrowth inhibition at concentration safe to normal quiescent cells (Table5). The data showed EC50 of compound 6 is in wide range of tumor lines100-600 nM which is 15-100 times more potent than sorafenib. Compound 6also demonstrated differentiated mechanism from PLX4032 and Mekinhibitors via inhibition of wildtype and mutant RAF.

TABLE 5 Potent Effects of compound 6 in RAF and Ras-mutated andnon-mutated tumor lines Mek Inhib. KG5 Sorafenib (UO126) Tissue OriginTumor Cell Line Aberration EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM) Melanoma CHL-1— 0.10 6.10 — Melanoma SK-MEL-2 N-Ras 0.14 14.27 — Melanoma SK-MEL-28B-RAF^(V600E) 0.40 17.60 — Melanoma 1205Lu B-RAF^(V600E) 0.83 — — BreastAC MDA-MB231 B-RAF/Ras 0.48 >10.00 — Colon Carcinoma CT26 — 0.71 — —Pancreatic AC XPA1-RFP K-Ras 0.28 — — Epithelial Carcinoma A431 HER10.43 — — Melanoma M21 — 0.45 — — Melanoma M21L — 0.42 — — Colon HCT116K-Ras 0.40 8.21 >20.0 Pancreatic (mouse R40P K-Ras 0.66 9.43 10.86spontaneous model) Breast MCF7 PI3K 0.68 >20.0 >20.0 Colon HT29B-RAF^(V600E) 0.62 >20.0 4.41

Example 21 Application of Compound 6 for Inhibition of Lymphangiogenesis

Lymphangiogenesis assays were performed by subcutaneously injecting 350μl of cold Matrigel containing saline or 1 mg/ml of VEGF-C(R&D Systems,Minneapolis, Minn.) into wild-type C57B1/6 mice. VEGF-C implanted micewere then treated by daily intraperitoneal injections of 200 μl twicedaily of saline, 5.25 mg/ml compound 6 or vehicle control (10% HS-15 and3.33% Dextrose) each day for 7 days, beginning with day 1 (n=7). After 7days, lymph nodes were removed, embedded in OCT, frozen and sectioned.Thin sections (5 μm) were fixed in ice cold acetone for five minutes,permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS),blocked in 8% normal donkey serum diluted in PBS for 1.5 hours at roomtemperature and then incubated with 2 μg/ml anti-murine Lyve-1(RDI-103PA50) in 8% normal donkey serum diluted in saline overnight at4° C. After extensive washing, slides were incubated with 1 g/mlcross-absorbed donkey anti-rabbit IgG (H+L) conjugated with Alexa Fluor488 (Invitrogen). Slides were counterstained with DAPI. Coverslips weremounted with Dako Cytomation fluorescent mounting medium (DakoCorporation, Carpinteria, Calif.). At least five microscopic fields pertissue section/mouse were analyzed for quantification studies. Resultsare shown in FIGS. 12A and 12B.

Example 22 Application of Compound 6 for Treatment of Fibrosis

Compound 6 (50 mg/Kg/day, oral) is evaluated for treatment of fibrosiswith 90 patients (two groups—45 patients with drug and 45 patients withplacebo) referred to the Interstitial Lund Disease clinic who isundergoing evaluation and or treatment for a new diagnosis of ILD. Thiscan include patients referred for presumed pulmonaryfibrosis/interstitial pneumonitis (IPF, UIP, NSIP), sarcoidosis,hypersensitivity pneumonitis, cryptogenic organising pneumonia,drug-induced, or other idiopathic ILDs. The patients are evaluated after6 months of treatment. Patients with treatment of compound 6 findimprovement over placebo group.

Example 23 The Growth Inhibitory Properties of Compound 6 on Tumor Cells

Compound 6 was profiled in the NCI60 panel by the NCI DevelopmentalTherapeutics Program and demonstrated an average growth inhibitoryconcentration (GI50) of 490 nM with potent growth inhibition across theentire panel of cell lines. See FIG. 13A. Additionally, compounds 6, 35,or 37 were compared to sorafenib, PLX 4720 or L779,450 in a cellproliferation assay. A549 cells, which endogenously express oncogenicK-RAS, were plated at 2500 cells/well of a 96-well plate in low serumDMEM (0.5% fetal bovine serum) overnight. The next morning, compoundswere serially diluted in DMSO and resuspended in fresh DMEM+0.5% medium,which was added to the cells for 72 h. At this time point the cells werefixed and proliferation was quantified using an ELISA forbromodeoxyuriding incorporation (BrdU ELISA, Millipore, Inc.). BrdUincorporation occurs during S phase and is the classical method fordetermining cell proliferation rates. In FIG. 14B-C, BrdU incorporationis expressed as a percentage of the BrdU incorporation occurring incells treated with 0.1% DMSO as control. A dotted line represents thegrowth rate of cells treated with DMSO alone. Compound 6, 35, or 37, allinhibitor cell proliferation (FIG. 13B), whereas the commerciallyavailable RAF inhibitors (sorafenib, PLX 4720, or L-779,450) all inducehyperproliferation when compared to the DMSO treated control cells.(FIG. 13C)

Example 24 Comparison of Compound 6 with Sorafenib RegardingPhosphorylation on CRAF S338

R40P cells are derived from a spontaneous pancreatic adenocarcinomamouse model which expresses K-ras^(G12D) while p16/Ink4a/ARF is lost.R40P cells were starved for 12 h and treated for 12 h with a MEKinhibitor (U0126), sorafenib, and compound 3 at 5 μM. A dose response of5, 2.5 and 1.25 μM was used for compound 6. At this time the cells werelysed and protein levels were quantified by BCA analysis. The lysateswere then loaded on an SDS-PAGE gel and transferred to nitrocellulosefor Western blotting. Immunoblotting was performed with the followingantibodies: pMEK1/2 S217/S221 (Cell Signaling), pCRAF S338 (CellSignaling), and total CRAF (BD Biosciences). Chemilluminescence was usedfor detection on film. It is important to note that all ATP-competitiveinhibitors we have utilized demonstrate a similar induction ofphosphorylation on CRAF S338. FIG. 14 shows that sorafenib induces anincrease in phosphorylation on CRAF S338, while Compound 6 does notpromote this increase.

Example 25 Cell Cycle and Confocal Microscopy Analysis of XPA-1 CellsTreated with the Allosteric RAF Inhibitor Compounds

Cell cycle and confocal microscopy analysis of XPA-1 cells treated withthe allosteric RAF inhibitor compound 6, the ATP-competitive inhibitorsorafenib, the MEK inhibitor (PD0325901), compound 3 or paclitaxel. Cellcycle analysis revealed that compound 6 treatment led to a G2/M arrestin all tumor cell lines tested, mimicking the activity of theanti-mitotic agent paclitaxel, while compound 3 a structural analogue ofcompound 6 that inhibits c-Kit, Flt-3 and PDGFR, but not RAF, showed noeffect. See FIGS. 15A and 15B. Importantly, treatment of cells withsorafenib or the MEK inhibitor PD0325901, led to ERK inhibition asexpected (not shown), yet had no effect on mitotic progression. For cellcycle analysis, cells were grown in complete DMEM+10% FBS in thepresence of each inhibitor and harvested after 20 h of inhibitortreatment. Cell cycle analysis was performed by flow cytometry followingpropidium iodide staining. The cells were harvested, fixed with ice coldmethanol, treated for 45 min with 10 μg/ml of RNAse and resuspended inPBS containing 10 μg/ml of PI, and analysed by flow cytometry. Cellcycle analysis was performed in cells treated with compound 6,sorafenib, or MEK inhibitor (PD0325901) at 5 μM concentration orpaclitaxel at 100 nM. For corresponding confocal microscopy images,cells attached to coverslips were fixed with cold methanol andpermeabilized in PBS containing 0.1% triton X-100 for two minutes andblocked for 60 minutes, at room temperature with 2% BSA in PBS. Cellswere stained with the DNA binding dye TOPRO-3 (Invitrogen) to label theDNA and laminB (Santa Cruz) to label the nuclear envelope. Theanti-Lamin B antibody was used at a 1:100 dilution for two hours at roomtemperature. After washing several times with PBS, cells were stainedfor two hours at room temperature, with secondary antibody diluted 1:300and co-incubated with TOPRO-3 (1:500) (Invitrogen). Samples were mountedin Vectashield hard-set mounting media (Vector Laboratories) and imagedon a Nikon Eclipse C1 confocal microscope with a 1.4 NA 60×oil-immersion lens, using minimum pinhole (30 μm). Images were capturedand processed using EZ-C1 3.50 imaging software (Nikon, Inc). Scalebars, 10 μm.

Example 26 Cell Cycle Analysis of Compound 6

Cell cycle analysis was performed by FACS. See FIG. 16. The graphrepresents the % of cells arrested in G2/M after treatment with 5 μMcompound 6 compared to 0.1% DMSO control. The graph depicts G2/Mquantification of human colon (HCT-116), pancreatic (Mia-Paca2, FG,XPA-1, BXPC3), breast (MB-MDA-231) and brain (U251) cancer cell lines.Error bars represent s.d. (n=4).

Example 27 Analysis of Compound 6 Against Melanoma Tumor Growth In Vivo

Human 1205 Lu melanoma cells, which express BRAF V600E, weresubcutaneously implanted into the flank of 6-8 week old female Nu/Numice. The tumor cells were trypsinized and resuspended in 1:1 PBS:growth factor depleted Matrigel (BD Biosciences) and 50 μl containing1×10̂6 cells was injected into the flank. An excess of mice bearingtumors were utilized and groups of 6 animals were chosen based on thesymmetry of the tumor and randomized to give each group an average of 50mm³ (day 10). Treatment was initiated at this point with vehicle or 50mg/kg of compound 6, bid, ip for 2 weeks (through day 24). The vehiclewas 10% HS-15 (known as Solutol, BASF, Inc.) in 3.33% dextrose. Tumorgrowth was monitored by measuring the tumor length and width withprecision calipers every 3 days and calculating tumor volume using thefollowing formula: Tumor volume=0.5*(length)*(width)², provided thesmaller measurement was utilized as the length. FIG. 17 demonstratesthat compound 6 suppresses melanoma tumor growth in vivo.

Example 28 Analysis of Compound 6 Against Breast Cancer Growth In Vivo

Tumors were generated by injection of MDA-MB231 human breast carcinomacells (1×10⁶ tumor cells in 50 μl of sterile PBS) into the mammaryfatpad of 6-8 week old female Nu/Nu mice. Tumors were allowed to growand the mice were randomized into two groups with an average tumorvolume of 100 mm³. Mice were dosed (bid, ip) with vehicle (10% HS-15,3.33% dextrose) or 50 mg/kg compound 6. Tumor growth was monitored bymeasuring the tumor length and width with precision calipers every 2days and calculating tumor volume using the following formula: Tumorvolume=0.5*(length)*(width)², provided the smaller measurement wasutilized as the length. In FIG. 18A, the MDA-MB-231 tumors were treatedfor three consecutive days, and the tumors were resected 1 h followingthe final vehicle or compound 6 dose and stained for phosphorylation onCRAF S338. Compound 6 clearly inhibits phosphorylation of S338 in thebreast tumors. This suggests that pS338 CRAF could serve as a potentialbiomarker for measuring activity of the drug in clinical settings. Scalebar 20 μM. FIG. 18B displays the tumor growth profile after treatmentwith compound 6 or vehicle. Compound 6 suppresses tumor growth ascompared to vehicle treatment. FIG. 18C shows the pS338 CRAF levels inthe MDA-MB231 cells in vitro. The cells were treated with DMSO (Ctrl),compound 6, or sorafenib at 5 μM for 6 h and cell lysates were resolvedon 10% SDS-PAGE and immunoblotting was performed with the followingantibodies: pS338 CRAF (Cell Signaling), CRAF (BD Pharmingen), and Actin(Sigma), all diluted 1:1000. Cells were lysed in RIPA buffer (50 mM TrispH 7.4, 100 mM NaCl, 0.1% SDS, 1.0% TritonX-100, 1% deoxycholate)supplemented with complete protease inhibitor mixture (Roche), 50 mMNaF, and 1 mM Na₃VO₄ and centrifuged at 13,000 g for 10 min at 4° C.Protein concentration was determined by BCA assay and 30 μg of proteinwas loaded per lane of 10% SDS-PAGE gel. Immunohistochemistry in FIG.18A was performed according to the manufacturer's recommendations(Vector Labs), on 5 μM sections of paraffin-embedded tumors from theorthotopic xenograft breast cancer mouse model (MDA-MB231). Forphospho-S338 CRAF immunohistochemistry, antigen retrieval was performedin citrate buffer pH 6.0 at 95° C. for 20 min. Sections were treatedwith 0.3% H₂O₂ for 30 min, blocked in normal goat serum, PBS-T for 30min followed by Avidin-D and then incubated overnight at 4° C. withprimary antibody against pS338 CRAF (Thermo Scientific) diluted 1:100 inblocking solution. Tissue sections were washed and then incubated withbiotinylated secondary antibody (1:500, Jackson ImmunoResearch) inblocking solution for 1 h. Sections were washed & incubated withVectastain ABC (Vector Labs) for 30 min. Staining was developed using aNickel-enhanced diamino-benzidine reaction (Vector Labs) and sectionswere counter-stained with hematoxylin.

Example 29 Cell Viability Assays

Tumor cells (A549 (FIG. 19A), T47D (FIG. 19B), and MDA-MB231 (FIG. 19C))were plated at 2500 cells/well of a 96-well plate in complete growthmedium (DMEM+10% FBS, antibiotic/antimycotic, fungizone, L-glutamine,sodium pyruvate, and non-essential amino acids). The cells were allowedto grow overnight and then serial dilutions of the correspondingcompounds in DMSO were added to fresh complete growth medium and addedto the cells for 72 h. Cell viability was measured by adding MTT (Sigma)at 5 mg/ml for 4 h to the cells and then removing the medium andresuspending each well in 50 μl DMSO to solubilize the crystals. The96-well plates were read on a plate-scanning spectrophotometer (BioTek)at an absorbance of 560 nm. The 11 pt cell viability curves were plottedusing GraphPad software and EC50s were calculated using the software andthe non-linear regression feature. Compounds such as compound 37 andcompound 35 demonstrate greatly improved potency relative to compound 6in each cell line tested—reaching single digit nM EC50s for inhibitingcell viability in some instances.

Example 30 Parenteral Composition

To prepare a parenteral pharmaceutical composition suitable foradministration by injection, 100 mg of a water-soluble salt of acompound 6 is dissolved in DMSO and then mixed with 10 mL of 0.9%sterile saline. The mixture is incorporated into a dosage unit formsuitable for administration by injection.

Example 31 Oral Composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of acompound 6 is mixed with 750 mg of starch. The mixture is incorporatedinto an oral dosage unit for, such as a hard gelatin capsule, which issuitable for oral administration.

Example 32 Sublingual (Hard Lozenge) Composition

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound 6 with 420 mg of powdered sugarmixed, with 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42mL mint extract. The mixture is gently blended and poured into a mold toform a lozenge suitable for buccal administration.

Example 33 Fast-Disintegrating Sublingual Tablet

A fast-disintegrating sublingual tablet is prepared by mixing 48.5% byweigh of a compound 6, 44.5% by weight of microcrystalline cellulose(KG-802), 5% by weight of low-substituted hydroxypropyl cellulose (50ÿm), and 2% by weight of magnesium stearate. Tablets are prepared bydirect compression (AAPS PharmSciTech. 2006; 7(2):E41). The total weightof the compressed tablets is maintained at 150 mg. The formulation isprepared by mixing the amount of compound of Formula (I) with the totalquantity of microcrystalline cellulose (MCC) and two-thirds of thequantity of low-substituted hydroxypropyl cellulose (L-HPC) by using athree dimensional manual mixer (lnversina®, Bioengineering AG,Switzerland) for 4.5 minutes. All of the magnesium stearate (MS) and theremaining one-third of the quantity of L-HPC are added 30 seconds beforethe end of mixing.

Example 34 Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound 6 is mixed with 50 mg of anhydrous citric acid and 100 mLof 0.9% sodium chloride solution. The mixture is incorporated into aninhalation delivery unit, such as a nebulizer, which is suitable forinhalation administration.

Example 35 Rectal Gel Composition

To prepare a pharmaceutical composition for rectal delivery, 100 mg of acompound 6 is mixed with 2.5 g of methylcelluose (1500 mPa), 100 mg ofmethylparaben, 5 g of glycerin and 100 mL of purified water. Theresulting gel mixture is then incorporated into rectal delivery units,such as syringes, which are suitable for rectal administration.

Example 36 Topical Gel Composition

To prepare a pharmaceutical topical gel composition, 100 mg of acompound 6 is mixed with 1.75 g of hydroxypropyl celluose, 10 mL ofpropylene glycol, 10 mL of isopropyl myristate and 100 mL of purifiedalcohol USP. The resulting gel mixture is then incorporated intocontainers, such as tubes, which are suitable for topicaladministration.

Example 37 Ophthalmic Solution Composition

To prepare a pharmaceutical opthalmic solution composition, 100 mg of acompound 6 is mixed with 0.9 g of NaCl in 100 mL of purified water andfiltered using a 0.2 micron filter. The resulting isotonic solution isthen incorporated into ophthalmic delivery units, such as eye dropcontainers, which are suitable for ophthalmic administration.

Example 38 Nasal Spray Solution

To prepare a pharmaceutical nasal spray solution, 10 g of a compound 6is mixed with 30 mL of a 0.05M phosphate buffer solution (pH 4.4). Thesolution is placed in a nasal administrator designed to deliver 100 ÿlof spray for each application.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A compound has the structure

or an N-oxide, N,N′-dioxide, N,N′,N″-trioxide, or a pharmaceuticallyacceptable salt thereof, wherein R₁ and R₂ are independently hydrogen,optional substituted alkyl, halogen, optional substituted amine, NH₂,optional substituted alkyoxy, optional substituted thioalkyl, CF₃S,optional substituted alkylsulfinyl or optional substitutedalkylsulfonyl; Z′ is N or C; R^(m) is C₁₋₆ alkyl, or halogen substitutedC₁₋₆ alkyl; and R₃ is independently a hydrogen, C₁₋₆ alkyl, halogensubstituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy, orC₃₋₁₀cycloalkyl; or, optionally, R^(m) and R₃ are joined to form a fiveto seven membered carbocycle; and n is 0-4.
 2. The compound of claim 1,wherein R₁ is optional substituted alkyoxy, optional substitutedthioalkyl, or CF₃S.
 3. The compound of claim 1, wherein R₂ is optionalsubstituted amine or NH₂.
 4. The compound of claim 1, wherein R₁ isoptional substituted alkyoxy or optional substituted thioalkyl and R₂ isNH₂.
 5. The compound of claim 1, wherein R^(m) is C₁₋₆ alkyl or CF₃. 6.The compound of claim 1, wherein R₃ is C₁₋₆ alkyl or halogen substitutedC₁₋₆ alkyl.
 7. The compound of claim 1, wherein R^(m) and R₃ are joinedto form a five to seven membered carbocycle.
 8. The compound of claim 1,wherein the compound is selected from


9. A process for preparing a compound of formula I:

comprising reacting a compound of formula II,

with a compound of formula (III),

in the presence of a base, wherein W is selected from the groupconsisting of pyridine, pyridazine, pyrimidine, pyrazine, triazine,14uinolone, isoquinoline, cinnoline, quinazoline, quinoxaline,1,2,4-benzotriazine and azaindole; X is O, S or NH; R₁ and R₂ areindependently a hydrogen, optional substituted alkyl, halogen, optionalsubstituted amine, —NH₂, —OH, optional substituted alkoxy, optionalsubstituted thioalkyl, —SCF₃, optional substituted alkylsulfinyl or anoptional substituted alkylsulfonyl; R₃ is independently a hydrogen, C₁₋₆alkyl, halogen substituted C₁₋₆ alkyl, halogen substituted C₁₋₆ alkoxy,or C₃₋₁₀cycloalkyl; or, optionally, two R3 are joined to form a 5 to 7membered carbocycle; and m is 1-5.
 10. A process for preparing acompound of formula Ia:

comprising reacting a compound of formula Iia,

with a compound of formula (IIIa),

in the presence of a base, wherein R₁ and R₂ are independently ahydrogen, optional substituted alkyl, halogen, optional substitutedamine, —NH₂, —OH, optional substituted alkoxy, optional substitutedthioalkyl, —SCF₃, optional substituted alkylsulfinyl or an optionalsubstituted alkylsulfonyl and R₃ is a hydrogen, halogen substituted C₁₋₆alkyl, or halogen substituted C₁₋₆ alkoxy.
 11. The process according toclaim 9, wherein the molar ratio of said compound of formula II to saidcompound of formula III is in the range from 0.6:1 to 0.9:1.
 12. Theprocess according to claim 9, wherein said base is an amine base. 13.The process according to claim 12, wherein said amine base is2,6-lutidine.
 14. The process according to claim 9, wherein R₁ is ahydrogen, —NH₂, optional substituted alkoxy, optional substitutedthioalkyl, CF₃S, optional substituted alkylsulfinyl or an optionalsubstituted alkylsulfonyl.
 15. The process according to claim 9, whereinR₂ is hydrogen, NH₂, or optional substituted alkoxy.
 16. The processaccording to claim 9, wherein R₁ and R₂ are independently an optionalsubstituted amine.
 17. The process according to claim 9, wherein R₁ isan optional substituted thioalkyl and R₂ is an optional substitutedamine.
 18. The process according to claim 17, wherein R₁ is a MeS and R₂is a NH₂.
 19. The process according to claim 18, wherein R₃ is ahydrogen.
 20. A process for preparing a compound of formula IV:

comprising reacting a compound of formula V,

with a compound of formula (VI),

in the presence of a base wherein L is a leaving group; Y is —SH, —OH,or —NH₂; R₁ and R₂ are independently hydrogen, optional substitutedalkyl, halogen, optional substituted protected amine, optionalsubstituted alkoxy, optional substituted thioalkyl, CF₃S, optionalsubstituted alkylsulfinyl or optional substituted alkylsulfonyl and R₄is a C₁₋₆ alkyl.
 21. The process according claim 20, wherein saidcompound of formula IV is a compound having the structure of formula IV:

said compound of formula V having the structure of Va,

said compound of formula VI is an alkyl hydroxybenzoate.
 22. The processaccording to claim 20, wherein R₁ and R₂ are independently an optionalsubstituted protected amine.
 23. The process according to claim 22,wherein said optional substituted protected amine is protected by anacid labile protecting group.
 24. The process according to claim 23,wherein R₁ and R₂ are independently a —NHBoc or N(Boc)₂.
 25. The processaccording to claim 20, wherein R₁ is an optional substituted thioalkyland R₂ is an optional substituted protected amine.
 26. The processaccording to claim 25, wherein R₁ is an —Sme and R₂ is a —N(Boc)₂. 27.The process according to claim 20, where said base is1,4-diazabicyclo[2.2.2]octane. 28.-63. (canceled)
 64. The compound ofclaim 1, wherein the compound is selected from

65.-71. (canceled)